EP1438068B1 - Antibody inhibitors of gdf-8 and uses thereof - Google Patents

Antibody inhibitors of gdf-8 and uses thereof Download PDF

Info

Publication number
EP1438068B1
EP1438068B1 EP02783984.4A EP02783984A EP1438068B1 EP 1438068 B1 EP1438068 B1 EP 1438068B1 EP 02783984 A EP02783984 A EP 02783984A EP 1438068 B1 EP1438068 B1 EP 1438068B1
Authority
EP
European Patent Office
Prior art keywords
antibody
gdf
seq
prt
antibodies
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Expired - Lifetime
Application number
EP02783984.4A
Other languages
German (de)
English (en)
French (fr)
Other versions
EP1438068A2 (en
EP1438068A4 (en
Inventor
Jane Aghajanian
Neil M. Wolfman
Geertruida M. Veldman
Monique V. Davies
Lisa-Anne Whittemore
Denise O'hara
Kristie Grove Bridges
Tejvir S. Khurana
Mary Bouxsein
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Wyeth LLC
Original Assignee
Wyeth LLC
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Wyeth LLC filed Critical Wyeth LLC
Priority to EP10183355A priority Critical patent/EP2316851A1/en
Publication of EP1438068A2 publication Critical patent/EP1438068A2/en
Publication of EP1438068A4 publication Critical patent/EP1438068A4/en
Application granted granted Critical
Publication of EP1438068B1 publication Critical patent/EP1438068B1/en
Anticipated expiration legal-status Critical
Expired - Lifetime legal-status Critical Current

Links

Images

Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/22—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against growth factors ; against growth regulators
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P11/00—Drugs for disorders of the respiratory system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P19/00—Drugs for skeletal disorders
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P19/00—Drugs for skeletal disorders
    • A61P19/08—Drugs for skeletal disorders for bone diseases, e.g. rachitism, Paget's disease
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P19/00—Drugs for skeletal disorders
    • A61P19/08—Drugs for skeletal disorders for bone diseases, e.g. rachitism, Paget's disease
    • A61P19/10—Drugs for skeletal disorders for bone diseases, e.g. rachitism, Paget's disease for osteoporosis
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P21/00—Drugs for disorders of the muscular or neuromuscular system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P21/00—Drugs for disorders of the muscular or neuromuscular system
    • A61P21/04—Drugs for disorders of the muscular or neuromuscular system for myasthenia gravis
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/02—Drugs for disorders of the nervous system for peripheral neuropathies
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • A61P3/02—Nutrients, e.g. vitamins, minerals
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • A61P3/04—Anorexiants; Antiobesity agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • A61P3/06—Antihyperlipidemics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • A61P3/08—Drugs for disorders of the metabolism for glucose homeostasis
    • A61P3/10—Drugs for disorders of the metabolism for glucose homeostasis for hyperglycaemia, e.g. antidiabetics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P5/00—Drugs for disorders of the endocrine system
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/475—Growth factors; Growth regulators
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2217/00—Genetically modified animals
    • A01K2217/07—Animals genetically altered by homologous recombination
    • A01K2217/075—Animals genetically altered by homologous recombination inducing loss of function, i.e. knock out
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/76—Antagonist effect on antigen, e.g. neutralization or inhibition of binding

Definitions

  • This invention relates to inhibitors of Growth Differentiation Factor-8 (GDF-8) proteins and methods of use for such inhibitors. More particularly, the invention provides novel antibodies and antibody fragments that are specifically reactive with GDF-8 proteins in vitro and in vivo.
  • the invention is particularly useful for diagnosing, preventing, or treating human or animal disorders in which an increase in muscle tissue would be therapeutically beneficial.
  • Exemplary disorders include neuromuscular disorders (e.g., muscular dystrophy and muscle atrophy), congestive obstructive pulmonary disease, muscle wasting syndrome, sarcopenia, and cachexia; adipose tissue disorders (e.g ., obesity); type 2 diabetes; and bone degenerative disease (e.g., osteoporosis).
  • GDF-8 Growth and Differentiation Factor-8
  • TGF- ⁇ Transforming Growth Factor-beta
  • TGF- ⁇ Transforming Growth Factor-beta
  • SGF- ⁇ Transforming Growth Factor-beta
  • GDF-8 is a negative regulator of skeletal muscle mass, and there is considerable interest in identifying factors which regulate its biological activity. For example, GDF-8 is highly expressed in the developing and adult skeletal muscle.
  • the GDF-8 null mutation in transgenic mice is characterized by a marked hypertrophy and hyperplasia of the skeletal muscle ( McPherron et al. (1997) Nature, 387: 83-90 ). Similar increases in skeletal muscle mass are evident in naturally occurring mutations of GDF-8 in cattle ( Ashmore et al. (1974) Growth, 38: 501-507 ; Swatland and Kieffer (1994) J. Anim. Sci., 38: 752-757 ; McPherron and Lee (1997) Proc. Natl. Acad. Sci. USA, 94: 124557-12461 ; and Kambadur et al. (1997) Genome Res., 7: 910-915 ).
  • GDF-8 is expressed in both developing and adult muscles, it is not clear whether it regulates muscle mass during development or in adults. Thus, the question of whether or not GDF-8 regulates muscle mass in adults is important from a scientific and therapeutic perspective. Recent studies have also shown that muscle wasting associated with HIV-infection in humans is accompanied by increases in GDF-8 protein expression ( Gonzalez-Cadavid at a/. (1998) PNAS, 95: 14938-43 ). In addition, GDF-8 can modulate the production of muscle-specific enzymes (e.g., creatine kinase) and modulate myoblast cell proliferation ( WO 00/43781 ).
  • muscle-specific enzymes e.g., creatine kinase
  • WO98/33887 discloses a transgenic GDF8 knockout mouse and shows that the transgenic mouse has increased muscle tissue. WO98/33887 also discloses polyclonal antibodies directed to murine GDF8.
  • a number of human and animal disorders are associated with loss or functional impairment of muscle tissue, including muscular dystrophy, muscle, atrophy, congestive obstructive pulmonary disease, muscle wasting syndrome, sarcopenia, and cachexia.
  • very few reliable or effective therapies exist for these disorders.
  • the serious symptoms associated with these disorders may be substantially reduced by employing therapies that increase the amount of muscle tissue in patients suffering from the disorders. While not curing the conditions, such therapies would significantly improve the quality of life for these patients and could ameliorate some of the effects of these diseases.
  • therapies that may contribute to an overall increase in muscle tissue in patients suffering from these disorders.
  • GDF-8 may also be involved in a number of other physiological processes, including glucose homeostasis in the development of type 2 diabetes and adipose tissue disorders, such as obesity.
  • GDF-8 modulates preadipocyte differentiation to adipocytes ( Kim et al. (2001) BBRC, 281: 902-906 ).
  • the GDF-8 protein is synthesized as a precursor protein consisting of an amino-terminal propeptide and a carboxy-terminal mature domain ( McPherron and Lee, (1997) Proc. Natl. Acad. Sci. USA, 94: 12457-12461 ). Before cleavage, the precursor GDF-8 protein forms a homodimer. The amino-terminal propeptide is then cleaved from the mature domain. The cleaved propeptide may remain noncovalently bound to the mature domain dimer, inactivating its biological activity ( Miyazono et al. (1988) J. Biol.
  • the propeptide is known as the "latency-associated peptide” (LAP), and the complex of mature domain and propeptide is commonly referred to as the "small latent complex” ( Gentry and Nash (1990) Biochemistry, 29: 6851-6857 ; Derynck et al. (1995) Nature, 316: 701-705 ; and Massague (1990) Ann. Rev. Cell Biol., 12: 597-641 ).
  • Other proteins are also known to bind to GDF-8 or structurally related proteins and inhibit their biological activity. Such inhibitory proteins include follistatin, and potentially, follistatin-related proteins ( Gamer et al. (1999) Dev. Biol., 208: 222-232 ).
  • the mature domain is believed to be active as a homodimer when the propeptide is removed.
  • GDF-8 is highly conserved in sequence and in function across species.
  • the amino acid sequence of murine and human GDF-8 is identical, as is the pattern of mRNA expression ( McPherron et al. (1997) Nature 387: 83-90 ; Gonzalez-Cadavid et al. (1998) Proc. Natl. Acad. Sci. USA 95: 14938-14943 ).
  • McPherron et al. (1997) Nature 387: 83-90 ; Gonzalez-Cadavid et al. (1998) Proc. Natl. Acad. Sci. USA 95: 14938-14943 ).
  • This conservation of sequence and function suggests that inhibition of GDF-8 in humans is likely to have a similar effect to inhibition of GDF-8 in mice.
  • GDF-8 is involved in the regulation of many critical biological processes. Due to its key function in these processes, GDF-8 may be a desirable target for therapeutic intervention.
  • therapeutic agents that inhibit the activity of GDF-8 may be used to treat human or animal disorders in which an increase in muscle tissue would be therapeutically beneficial, particularly muscle and adipose tissue disorders, bone degenerative diseases, neuromuscular disorders, and diabetes, as discussed above.
  • the present invention provides novel protein inhibitors comprising antibodies and antibody fragments that are specifically reactive with a mature GDF-8 protein, whether it is in a monomeric form, active dimeric form, or complexed in the GDF-8 latent complex.
  • the antibodies bind to an epitope on the mature GDF-8 protein, which results in a reduction in one or more of the biological activities associated with GDF-8, relative to a mature GDF-8 protein that is not bound by the same antibody.
  • the presently disclosed antibodies reduce GDF-8 activity associated with negative regulation of skeletal muscle mass and/or bone density.
  • the presently disclosed antibodies possess unique and unexpected biological properties. For instance, one of skill in the art would typically expect good neutralizing antibodies to strongly bind to the active GDF-8 protein in vitro, forming a stable inhibitory complex with the protein.
  • a neutralizing antibody also called an inhibitory antibody, having a high affinity for a particular protein will typically be expected to provide higher levels of neutralization relative to a lower affinity antibody to the same protein.
  • the present inventors have discovered antibodies that only weakly bind to and neutralize active GDF-8 protein in vitro, yet are effective in vivo. The discovery of such antibodies led, in turn, to the identification of a specific site on GDF-8 to which the antibodies bind. It is therefore expected that any antibody specifically binding to the identified site would similarly possess in vivo neutralizing properties.
  • the presently disclosed antibodies possess unique and unexpected properties.
  • the antibodies not only recognize mature GDF-8 protein in its monomeric and dimeric forms, but also recognize the intact GDF-8 latent complex.
  • the presently disclosed antibodies may be administered in a therapeutically effective dose to treat or prevent medical conditions in which an increase in muscle tissue mass or bone density would be therapeutically beneficial.
  • Diseases and disorders that may be treated by these GDF-8 antibodies include muscle or neuromuscular disorders such as muscular dystrophy, muscle atrophy, congestive obstructive pulmonary disease, muscle wasting syndrome, sarcopenia, and cachexia; adipose tissue disorders such as obesity; metabolic disorders such as type 2 diabetes, impaired glucose tolerance, metabolic syndromes ( e.g. , syndrome X), insulin resistance induced by trauma ( e.g ., burns); and bone degenerative disease such as osteoporosis, especially in the elderly and/or postmenopausal women.
  • GDF-8 antibodies Additional metabolic bone diseases and disorders amenable to treatment with these GDF-8 antibodies include low bone mass due to chronic glucocorticoid therapy, premature gonadal failure, androgen suppression, vitamin D deficiency, secondary hyperparathyroidism, nutritional deficiencies, and anorexia nervosa.
  • the presently disclosed antibodies may be used as a diagnostic tool to quantitatively or qualitatively detect mature GDF-8 protein or fragments thereof, regardless of whether it is in a monomeric form, dimeric active form, or complexed in the GDF-8 latent complex.
  • the antibodies may be used to detect quantitatively or qualitatively mature GDF-8 protein in a cell, bodily fluid, tissue, or an organ. The presence or amount of mature GDF-8 protein detected is then correlated with one or more of the medical conditions listed above.
  • the presently disclosed antibodies may be provided in a diagnostic kit.
  • the kit may contain other components that aid the detection of mature GDF-8 protein, and help correlate the results with one or more of the medical conditions described above.
  • Figure 3 mature GDF-8 AA sequence 16
  • Figure 3 BMP-11 AA sequence 18 JA-16 eptitope region from GDF-8 17-64
  • Figure 6A Overlapping 13-mer peptides corresponding to portions of the GDF-8 sequence 65 Biotinylated N-terminal peptide derived from GDF-8 66-104, 106-113, 115-128 Mutated versions of SEQ ID NO:18 130 AA sequence of GDF-8 propeptide (accession no. xp 030768) 131 AA sequence of BMP-11 propeptide (accession no. xp 049170)
  • antibody refers to one or more polyclonal antibodies, monoclonal antibodies, antibody compositions, antibodies having mono- or poly-specificity, humanized antibodies, single-chain antibodies, chimeric antibodies, CDR-grafted antibodies, antibody fragments such as Fab, F(ab') 2 , Fv, and other antibody fragments which retain the antigen binding function of the parent antibody.
  • chimeric antibodies refers to molecules in which a portion of the heavy and/or light chain is identical or homologous to corresponding sequences from a particular species (or belonging to a particular antibody class or subclass), while the remainder of the chain(s) is identical or homologous to corresponding sequences derived from a different species (or belonging to a different antibody class or subclass).
  • Such chimeric antibodies are described by Morrison, et al. (1984) Proc. Natl. Acad. Sci. USA 81: 6851-6855 .
  • epitope refers to a molecule or portion of a molecule that is capable of specifically reacting with an anti-GDF-8 monoclonal antibody. Epitopes may comprise proteins, protein fragments, peptides, carbohydrates, lipids, or other molecules, but are most commonly proteins, short oligopeptides, or combinations thereof.
  • GDF polypeptide and “GDF protein” refer generally to any growth and differentiation factors that are structurally or functionally related to GDF-8.
  • GDF inhibitor includes any agent capable of inhibiting activity, expression, processing, or secretion of a GDF protein.
  • Such inhibitors include proteins, antibodies, peptides, peptidomimetics, ribozymes, anti-sense oligonucleotides, double-stranded RNA, and other small molecules which specifically inhibit the GDF proteins.
  • GDF-8 or GDF-8 protein refer to a specific growth and differentiation factor. The terms include the full length unprocessed precursor form of the protein, as well as the mature and propeptide forms resulting from post-translational cleavage. The terms also refer to any fragments of GDF-8 that maintain the known biological activities associated with the protein, as discussed herein, including sequences that have been modified with conservative or non-conservative changes to the amino acid sequence.
  • GDF-8 molecules may be derived from any source, natural or synthetic.
  • the human form of mature GDF-8 protein is provided in SEQ ID NO:15.
  • the present invention also encompasses GDF-8 molecules from all other sources, including GDF-8 from bovine, chicken, murine, rat, porcine, ovine, turkey, baboon, and fish. These various GDF-8 molecules have been described in McPherron et al. (1997) Proc. Natl. Acad. Sci. USA, 94: 12457-12461 .
  • GDF-8 refers to the protein that is cleaved from the carboxy-terminal domain of the GDF-8 precursor protein.
  • the mature GDF-8 may be present as a monomer, homodimer, or in a GDF-8 latent complex. Depending on the in vivo or in vitro conditions, an equilibrium between any or all of these different forms may exist. GDF-8 is believed to be biologically active as homodimer. In its biologically active form, the mature GDF-8 is also referred to as "active GDF-8.”
  • GDF-8 propeptide refers to the polypeptide that is cleaved from the amino-terminal domain of the GDF-8 precursor protein.
  • the GDF-8 propeptide is capable of binding to the propeptide binding domain on the mature GDF-8.
  • GDF-8 latent complex refers to the complex of proteins formed between the mature GDF-8 homodimer and the GDF-8 propeptide. It is believed that two GDF-8 propeptides associate with a GDF-8 homodimer to form an inactive tetrameric complex.
  • the latent complex may include other GDF-8 inhibitors in place of or in addition to one or more of the GDF-8 propeptides.
  • GDF-8 inhibitor includes any agent capable of inhibiting the activity, expression, processing, or secretion of GDF-8 protein.
  • inhibitors include proteins, antibodies, peptides, peptidomimetics, ribozymes, anti-sense oligonucleotides, double-stranded RNA, and other small molecules that specifically inhibit the activity of GDF-8 protein.
  • Such inhibitors are said to "neutralize” or “reduce” the biological activity of GDF-8 protein.
  • GDF-8 activity refers to one or more of growth-regulatory or morphogenetic activities associated with active GDF-8 protein.
  • active GDF-8 is a negative regulator of skeletal muscle.
  • Active GDF-8 can also modulate the production of muscle-specific enzymes (e.g., creatine kinase), stimulate myoblast cell proliferation, and modulate preadipocyte differentiation to adipocytes.
  • isolated refers to a molecule that is substantially free of its natural environment.
  • an isolated protein is substantially free of cellular material or other contaminating proteins from the cell or tissue source from which it is derived.
  • substantially free of cellular material refers to preparations where the isolated protein is at least 70% to 80% (w/w) pure, optionally at least 80%-89% (w/w) pure, optionally 90-95% pure; and optionally at least 96%, 97%, 98%, 99% or 100% (w/w) pure.
  • mammal for purposes of treatment refers to any animal classified as a mammal, including humans, domestic and farm animals, and zoo, sports, or pet animals, such as dogs, horses, cats, sheep, pigs, cows, etc.
  • the mammal is human in one embodiment of the invention.
  • the term "monoclonal antibody” refers to one or more antibodies from a substantially homogeneous antibody population that is directed against a single antigenic epitope.
  • the term encompasses humanized antibodies, single-chain antibodies, chimeric antibodies, CDR-grafted antibodies, antibody fragments such as Fab, F(ab') 2 , Fv, and other antibody fragments which retain the antigen binding function of the parent antibody.
  • monoclonal antibody is not limited to any particular species or source of the antibody, or the manner by which it is made.
  • Monoclonal antibodies may be made via traditional hybridoma techniques ( Kohler and Milstein (1975) Nature, 256: 495-499 ), recombinant DNA methods ( U.S. Pat. No. 4,816,567 ), or phage antibody libraries ( Clackson et al. (1991) Nature, 352: 624-628 ; Marks et al. (1991) J. Mol. Biol., 222: 581-597 ).
  • Monoclonal antibodies of any mammalian or non-mammalian species can be used in this invention.
  • the antibodies may be derived from primates (e.g ., human, orangutan, etc.), avian (e.g ., chicken, turkey, etc.), bovine, murine, rat, porcine, ovine, or fish.
  • the antibodies are of rat, murine, or human origin.
  • neutralize and “neutralizing” refer to a reduction in the activity of GDF-8 by a GDF-8 inhibitor, relative to the activity of a GDF-8 molecule that is not bound by the same inhibitor.
  • a “neutralizing” antibody reduces the activity of GDF-8 relative to the activity of a GDF-8 molecule not bound by the same antibody.
  • the activity of the GDF-8 protein, when bound by one or more of the presently disclosed GDF-8 inhibitors is reduced at least about 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, or 55%, optionally at least about 60%, 62%, 64%, 66%, 68%, 70%, 72%, 72%, 76%, 78%, 80%, 82%, 84%, 86%, or 88%, optionally at least about 90%, 91%, 92%, 93%, or 94%, and optionally at least 95% to 100% relative to a GDF-8 protein that is not bound by one or more of the presently disclosed GDF-8 inhibitors.
  • the presently disclosed GDF-8 inhibitors e.g ., the presently disclosed antibodies
  • binding means that two molecules form a complex that is relatively stable under physiologic conditions.
  • an antigen-binding domain is specific for a particular epitope, which is carried by a number of antigens, in which case the specific binding member carrying the antigen-binding domain will be able to bind to the various antigens carrying the epitope.
  • Specific binding is characterized by a high affinity and a low to moderate capacity. Nonspecific binding usually has a low affinity with a moderate to high capacity. Typically, the binding is considered specific when the affinity constant K a is higher than 10 6 M -1 , or can be higher than 10 8 M -1 .
  • non-specific binding can be reduced without substantially affecting specific binding by varying the binding conditions.
  • Such conditions are known in the art, and a skilled artisan using routine techniques can select appropriate conditions.
  • the conditions are usually defined in terms of concentration of antibodies, ionic strength of the solution, temperature, time allowed for binding, concentration of non-related molecules (e.g., serum albumin, milk casein), etc. Exemplary conditions are set forth in Example 4.
  • TGF- ⁇ superfamily refers to a family of structurally -related growth factors, all of which are endowed with physiologically important growth-regulatory and morphogenetic properties. This family of related growth factors is well known in the art ( Kingsley et al. (1994) Genes Dev., 8: 133-146 ; Hoodless et al. (1998) Curr. Topics Microbiol. Immunol., 228: 235-272 ).
  • the TGF- ⁇ superfamily includes Bone Morphogenetic Proteins (BMPs), Activins, Inhibins, Mullerian Inhibiting Substance, Glial-Derived.
  • GDFs Growth and Differentiation Factors
  • BMP-11 Growth and Differentiation Factors
  • treatment refers to both therapeutic treatment and prophylactic or preventative measures.
  • Those in need of treatment may include individuals already having a particular medical disorder as well as those who may ultimately acquire the disorder ( i.e ., those needing preventative measures).
  • Intact antibodies are usually heterotetrameric glycoproteins of about 150,000 daltons, composed of two identical light (L) chains and two identical heavy (H) chains. Each light chain is linked to a heavy chain by one covalent disulfide bond, while the number of disulfide linkages varies among the heavy chains of different immunoglobulin isotypes. Each heavy and light chain also has regularly spaced intrachain disulfide bridges. Each heavy chain has at one end a variable domain (V H ) followed by a number of constant domains.
  • V H variable domain
  • Each light chain has a variable domain at one end (V L ) and a constant domain at its other end; the constant domain of the light chain is aligned with the first constant domain of the heavy chain, and the light-chain variable domain is aligned with the variable domain of the heavy chain.
  • Particular amino acid residues are believed to form an interface between the light- and heavy-chain variable domains ( Clothia et al. (1985) J. Mol. Biol., 186: 651-663 ; Novotny and Haber (1985) Proc. Natl. Acad. Sci. USA, 82: 4592-4596 ).
  • immunoglobulins can be assigned to different classes. There are five major classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, and several of these may be further divided into subclasses (isotypes), e.g., IgA1 and IgA2 for IgA; IgGI, IgG2, IgG3, IgG4 for IgG in humans, and IgG1, IgG2a, IgG2b, and IgG3 for IgG in mouse.
  • the heavy-chain constant domains that correspond to the major classes of immunoglobulins are called ⁇ , ⁇ , ⁇ , ⁇ , and ⁇ , respectively.
  • the subunit structures and three-dimensional configurations of different classes of immunoglobulins are well known in the art.
  • each light chain is composed of an N-terminal variable (V) domain (V L ) and a constant (C) domain (C L ).
  • V L N-terminal variable domain
  • C L constant domain
  • Each heavy chain is composed of an N-terminal V domain, three or four C domains, and a hinge region.
  • the C H domain most proximal to V H is designated as C H 1.
  • the V H and V L domain consist of four regions of relatively conserved sequence called framework regions (FR1, FR2, FR3, and FR4), which form a scaffold for three regions of hypervariable sequence (complementarity determining regions, CDRs).
  • CDRs contain most of the residues responsible for specific interactions with the antigen.
  • CDRs are referred to as CDR1, CDR2, and CDR3.
  • CDR constituents on the on the heavy chain are referred to as H1, H2, and H3, while CDR constituents on the light chain are referred to as L1, L2, and L3.
  • CDR3 is the greatest source of molecular diversity within the antibody-binding site.
  • H3, for example can be as short as two amino acid residues or greater than 26.
  • the locations of immunoglobulin variable domains in a given antibody may be determined as described, for example, in Sequences of Proteins of Immunological Interest, US Department of Health and Human Services, eds. Kabat et al., 1991 .
  • Antibody diversity is created by the use of multiple germline genes encoding variable regions and a variety of somatic events.
  • the somatic events include recombination of variable gene segments with diversity (D) and joining (J) gene segments to make a complete V H region and the recombination of variable and joining gene segments to make a complete V L region.
  • the recombination process itself is imprecise, resulting in the loss or addition of amino acids at the V(D)J junctions.
  • Antibodies may be raised against any portion of a protein which provides an antigenic epitope.
  • the presently disclosed antibodies bind to a mature GDF-8 protein as set forth in SEQ ID NO:15; between amino acid 1 and amino acid 50; optionally between amino acid 1 and amino acid 25; and optionally between amino acid 1 and 15 of SEQ ID NO:15.
  • the presently disclosed antibodies specifically bind to the sequence Asp-Glu-His-Xaa-Thr (SEQ ID NO:2) in any one of the proteins belonging to the TGF- ⁇ superfamily, where Xaa is Ala, Gly, His, Met, Asn, Arg, Ser, Thr, or Trp.
  • the antibodies specifically bind to the peptide sequence Asp-Glu-His-Xaa-Thr (SEQ ID NO:2) in GDF-8, where Xaa is Ala, Gly, His, Met, Asn, Arg, Ser, Thr, or Trp.
  • the antibodies specifically bind to Asp-Glu-His-Ser-Thr (SEQ ID NO:3) in the mature GDF-8 protein (SEQ ID NO:15).
  • the presently disclosed antibodies specifically bind to the peptide sequence Asp-Phe-Gly-Leu-Asp-Cys-Asp-Glu-His-Xaa-Thr-Gfu-Ser-Arg-Cys (SEQ ID NO:5) in any one of the proteins belonging to the TGF- ⁇ superfamily, where Xaa is Ala, Gly, His, Met, Asn, Arg, Ser, Thr, or Trp.
  • the antibodies specifically bind to the peptide sequence Asp-Phe-Gly-Leu-Asp-Cys-Asp-Glu-His-Xaa-Thr-Glu-Ser-Arg-Cys (SEQ ID NO:5) in GDF-8, where Xaa is Ala, Gly, His, Met, Asn, Arg, Ser, Thr, or Trp.
  • the antibodies specifically bind to the peptide sequence Asp-Phe-Gly-Leu-Asp-Cys-Asp-Glu-His-Ser-Thr-Glu-Ser-Arg-Cys (SEQ ID NO:8) in the mature GDF-8 protein (SEQ ID NO:15).
  • the GDF-8 protein to which the presently disclosed antibodies may specifically bind is optionally at least about 75%-80% identical to SEQ ID NO:15, optionally at least about 81% to about 85% identical to SEQ ID NO:15, optionally at least about 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, or 94% identical to SEQ ID NO:15, and optionally at least about 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO:15.
  • the GDF-8 protein optionally comprises SEQ ID NO:15.
  • the presently disclosed antibodies may specifically bind to the BMP-11 protein.
  • the BMP-11 protein is optionally at least about 75%-80% identical to SEQ ID NO:16, optionally at least about 81% to about 85% identical to SEQ ID NO:16, optionally at least about 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, or 94% identical to SEQ ID NO:16, and optionally at least about 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO:16.
  • the BMP-11 protein optionally comprises SEQ ID NO:16.
  • the antibody comprises the amino acid sequence of SEQ ID NO: 1 as a part of the variable region of the heavy chain.
  • the antibody comprises at least one single chain CDR chosen from the amino acids 30-35 of SEQ ID NO:1, amino acids 50-66 of SEQ ID NO:1, and amino acids 99-102 of SEQ ID NO:1.
  • the antibodies of the invention may contain any number of conservative or non-conservative changes to their respective amino acid sequences without altering their biological properties. Changes can be made in either the framework (FR) or in the CDR regions. While changes in the framework regions are usually designed to improve stability and immunogenicity of the antibody, changes in the CDRs are usually designed to increase affinity of the antibody for its target. Such affinity-increasing changes are typically determined empirically by altering the CDR region and testing the antibody. Such alterations can be made according to the methods described in Antibody Engineering, 2nd. ed., Oxford University Press, ed. Borrebaeck, 1995 .
  • Conservative amino acid modifications are based on the relative similarity of the amino acid side-chain substituents, for example, their hydrophobicity, hydrophilicity, charge, size, and the like.
  • Exemplary conservative substitutions which take various of the foregoing characteristics into consideration are well known to those of skill in the art and include: arginine and lysine; glutamate and aspartate; serine and threonine; glutamine and asparagine; and valine, leucine, and isoleucine. Further details on such changes are described in the following sections. Unlike in CDRs, more substantial non-conservative changes in structure framework regions (FRs) can be made without adversely affecting the binding properties of an antibody.
  • FRs structure framework regions
  • Changes to FRs include, but are not limited to, humanizing a non-human derived framework or engineering certain framework residues that are important for antigen contact or for stabilizing the binding site, e.g., changing the class or subclass of the constant region, changing specific amino acid residues which might after an effector function such as Fc receptor binding ( Lund et al. (1991) J. Immun. 147: 2657-2662 and Morgan et al. (1995) Immunology 86: 319-324 ), or changing the species from which the constant region is derived as described below.
  • humanizing a non-human derived framework or engineering certain framework residues that are important for antigen contact or for stabilizing the binding site e.g., changing the class or subclass of the constant region, changing specific amino acid residues which might after an effector function such as Fc receptor binding ( Lund et al. (1991) J. Immun. 147: 2657-2662 and Morgan et al. (1995) Immunology 86: 319-324 ), or
  • the presently disclosed antibodies specifically bind to mature GDF-8 protein, regardless of whether it is in monomeric form, active dimer form, or complexed in a GDF-8 latent complex, with an affinity of between about 10 6 and about 10 11 M -1 , optionally between about 10 8 and about 10 11 M -1 .
  • the antibodies of the present disclosure may comprise polyclonal antibodies, monoclonal antibodies, antibody compositions, antibodies having mono- or poly-specificity, humanized antibodies, single-chain antibodies, CDR-grafted antibodies, antibody fragments such as Fab, F(ab') 2 , Fv, and other antibody fragments which retain the antigen binding function of the parent antibody.
  • the presently disclosed antibodies may also be modified to chimeric antibodies. For instance, a human Fc region can be fused to a GDF-8 binding region from a murine antibody to generate a chimeric antibody. By replacing other portions of the murine antibody (outside of the antigen binding region) with corresponding human antibody fragments, a humanized antibody may be produced.
  • Such chimeric or humanized antibodies may display enhanced biological specificity or in vivo stability. They are particularly useful in designing antibodies for human therapies. It is understood that practitioners are familiar with the standard resource materials which describe specific conditions and procedures for the construction, manipulation, production, and isolation of antibodies (see, for example, Harlow and Lane (1988) Antibodies: A Laboratory Manual, Cold Spring Harbor Press, Cold Spring Harbor, New York ).
  • the present disclosure also provides cells, such as hybridomas, that produce any of the presently disclosed antibodies.
  • One of skill in the art is familiar with the many cells that are suitable for producing antibodies. Any cell compatible with the present invention may be used to produce the presently disclosed antibodies.
  • the presently disclosed antibodies are produced by a hybridoma cell.
  • a hybridoma cell line, which produces murine anti-GDF-8 JA-16 antibody has been deposited with American Tissue Culture Collection (Deposit Designation Number PTA-4236) on April 18, 2002. The address of the depository is 10801 University Boulevard, Manassas, VA 20110.
  • the antibodies of the present disclosure are useful to prevent, diagnose, or treat various medical disorders in humans or animals.
  • the antibodies are used to inhibit or reduce one or more activities associated with the GDF protein, relative to a GDF protein not bound by the same antibody.
  • the antibodies inhibit or reduce one or more of the activities of mature GDF-8 (regardless of whether in monomeric form, active dimeric form, or complexed in a GDF-8 latent complex) relative to a mature GDF-8 protein that is not bound by the same antibodies.
  • the activity of the mature "GDF-8 protein, when bound by one or more of the presently disclosed antibodies, is inhibited at least 50%, optionally at least 60, 62, 64, 66, 68, 70, 72, 72, 76, 78, 80, 82, 84, 86, or 88%, optionally at least 90, 91, 92, 93, or 94%, and optionally at least 95% to 100% relative to a mature GDF-8 protein that is not bound by one or more of the presently disclosed antibodies.
  • the medical disorder being diagnosed, treated, or prevented by the presently disclosed antibodies is optionally a muscle and neuromuscular disorder; an adipose tissue disorder such as obesity; type 2 diabetes, impaired glucose tolerance, metabolic syndromes (e.g., syndrome X), insulin resistance induced by trauma such as burns; or bone degenerative disease such as osteoporosis.
  • a muscle and neuromuscular disorder such as obesity
  • type 2 diabetes impaired glucose tolerance
  • metabolic syndromes e.g., syndrome X
  • insulin resistance induced by trauma such as burns
  • bone degenerative disease such as osteoporosis.
  • the medical condition is optionally a muscle or neuromuscular disorder, such as muscular dystrophy, muscle atrophy, congestive obstructive pulmonary disease, muscle wasting syndrome, sarcopenia, or cachexia and disorders associated with a loss of bone, which include osteoporosis, especially in the elderly and/or postmenopausal women, glucocorticoid-induced osteoporosis, osteopenia, and osteoporosis-related fractures.
  • a muscle or neuromuscular disorder such as muscular dystrophy, muscle atrophy, congestive obstructive pulmonary disease, muscle wasting syndrome, sarcopenia, or cachexia and disorders associated with a loss of bone, which include osteoporosis, especially in the elderly and/or postmenopausal women, glucocorticoid-induced osteoporosis, osteopenia, and osteoporosis-related fractures.
  • GDF-8 antibodies of the invention include low bone mass due to chronic glucocorticoid therapy, premature gonadal failure, androgen suppression, vitamin D deficiency, secondary hyperparathyroidism, nutritional deficiencies, and anorexia nervosa.
  • the antibodies are optionally used to prevent, diagnose, or treat such medical disorders in mammals, optionally in humans.
  • an "effective amount" of the antibody is a dosage which is sufficient to reduce the activity of GDF proteins to achieve a desired biological outcome (e.g., increasing muscle mass or strength).
  • a therapeutically effective amount may vary with the subject's age, condition, and sex, as well as the severity of the medical condition in the subject.
  • the dosage may be determined by an physcian and adjusted, as necessary, to suit observed effects of the treatment.
  • the compositions are administered so that antibodies are given at a dose between 1 ⁇ g/kg and 20 mg/kg.
  • the antibodies are given as a bolus dose, to maximize the circulating levels of antibodies for the greatest length of time after the dose. Continuous infusion may also be used after the bolus dose.
  • the methods of treating, diagnosing, or preventing the above medical conditions with the presently disclosed antibodies can also be used on other proteins in the TGF- ⁇ superfamily. Many of these proteins, e.g., BMP-11, are related in structure to GDF-8. Accordingly, in another embodiment, the invention provides methods of treating the aforementioned disorders by administering to a subject an antibody capable of inhibiting BMP-11 or activin, either alone or in combination with other TGF- ⁇ inhibitors, such as a neutralizing antibody against GDF-8.
  • the antibodies of the present invention may be used to detect the presence of proteins belonging to the TGF- ⁇ superfamily, such as BMP-11 and GDF-8. By correlating the presence or level of these proteins with a medical condition, one of skill in the art can diagnose the associated medical condition.
  • the medical conditions that may be diagnosed by the presently disclosed antibodies are set forth above.
  • Such detection methods are well known in the art and include ELISA, radioimmunoassay, immunoblot, western blot, immunofluorescence, immuno-precipitation, and other comparable techniques.
  • the antibodies may further be provided in a diagnostic kit that incorporates one or more of these techniques to detect a protein (e.g., GDF-8).
  • a kit may contain other components, packaging, instructions, or other material to aid the detection of the protein and use of the kit.
  • the antibodies are intended for diagnostic purposes, it may be desirable to modify them, for example with a ligand group (such as biotin) or a detectable marker group (such as a fluorescent group, a radioisotope or an enzyme).
  • a ligand group such as biotin
  • a detectable marker group such as a fluorescent group, a radioisotope or an enzyme.
  • the antibodies may be labeled using conventional techniques. Suitable labels include fluorophores, chromophores, radioactive atoms, electron-dense reagents, enzymes, and ligands having specific binding partners. Enzymes are typically detected by their activity.
  • horseradish peroxidase is usually detected by its ability to convert 3,3',5,5'-tetramethylbenzidine (TMB) to a blue pigment, quantifiable with a spectrophotometer.
  • TMB 3,3',5,5'-tetramethylbenzidine
  • suitable labels include, for example, one of the binding partners such as biotin and avidin or streptavidin, IgG and protein A, and various receptor-ligand couples known in the art.
  • Other permutations and possibilities will be readily apparent to those of ordinary skill in the art, and are considered as equivalents within the scope of the instant invention.
  • compositions comprising the presently disclosed antibodies. Such compositions may be suitable for pharmaceutical use and administration to patients.
  • the compositions typically comprise one or more antibodies of the present invention and a pharmaceutically acceptable excipient.
  • pharmaceutically acceptable excipient includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like, that are compatible with pharmaceutical administration. The use of such media and agents for pharmaceutically active substances is well known in the art.
  • the compositions may also contain other active compounds providing supplemental, additional, or enhanced therapeutic functions.
  • the pharmaceutical compositions may also be included in a container, pack, or dispenser together with instructions for administration.
  • a pharmaceutical composition of the invention is formulated to be compatible with its intended route of administration. Methods to accomplish the administration are known to those of ordinary skill in the art. It may also be possible to obtain compositions which may be topically or orally administered, or which may be capable of transmission across mucous membranes.
  • the administration may, for example, be intravenous, intraperitoneal, intramuscular, intracavity, subcutaneous or transdermal.
  • Solutions or suspensions used for intradermal or subcutaneous application typically include one or more of the following components: a sterile diluent such as water for injection, saline solution, fixed oils, polyethylene glycols, glycerine, propylene glycol or other synthetic solvents; antibacterial agents such as benzyl alcohol or methyl parabens; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid; buffers such as acetates, citrates or phosphates; and agents for the adjustment of tonicity such as sodium chloride or dextrose.
  • the pH can be adjusted with acids or bases, such as hydrochloric acid or sodium hydroxide.
  • Such preparations may be enclosed in ampoules, disposable syringes or multiple dose vials made of glass or plastic.
  • compositions suitable for injection include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersion.
  • suitable carriers include physiological saline, bacteriostatic water, Cremophor ELTM (BASF, Parsippany, NJ) or phosphate buffered saline (PBS).
  • the composition must be sterile and should be fluid to the extent that easy syringability exists. It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms such as bacteria and fungi.
  • the carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyetheylene glycol, and the like), and suitable mixtures thereof.
  • the proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants.
  • Prevention of the action of microorganisms can be achieved by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, and the like.
  • isotonic agents for example, sugars, polyalcohols such as manitol, sorbitol, sodium chloride will be included in the composition.
  • Prolonged absorption of the injectable compositions can be brought about by including in the composition an agent which delays absorption, for example, aluminum monostearate and gelatin.
  • Oral compositions generally include an inert diluent or an edible carrier. They can be enclosed in gelatin capsules or compressed into tablets. For the purpose of oral therapeutic administration, the antibodies can be incorporated with excipients and used in the form of tablets, troches, or capsules. Pharmaceutically compatible binding agents, and/or adjuvant materials can be included as part of the composition.
  • the tablets, pills, capsules, troches, and the like can contain any of the following ingredients, or compounds of a similar nature; a binder such as microcrystalline cellulose, gum tragacanth or gelatin; an excipient such as starch or lactose, a disintegrating agent such as alginic acid, Primogel, or corn starch; a lubricant such as magnesium stearate or Sterotes; a glidant such as colloidal silicon dioxide; a sweetening agent such as sucrose or saccharin; or a flavoring agent such as peppermint, methyl salicylate, or orange flavoring.
  • a binder such as microcrystalline cellulose, gum tragacanth or gelatin
  • an excipient such as starch or lactose, a disintegrating agent such as alginic acid, Primogel, or corn starch
  • a lubricant such as magnesium stearate or Sterotes
  • a glidant such as colloidal silicon dioxide
  • the antibodies are delivered in the form of an aerosol spray from pressured container or dispenser which contains a suitable propellant, e.g., a gas such as carbon dioxide, or a nebulizer.
  • a suitable propellant e.g., a gas such as carbon dioxide, or a nebulizer.
  • Systemic administration can also be by transmucosal or transdermal means.
  • penetrants appropriate to the barrier to be permeated are used in the formulation.
  • penetrants are generally known in the art, and include, for example, for transmucosal administration, detergents, bile salts, and fusidic acid derivatives.
  • Transmucosal administration can be accomplished through the use of nasal sprays or suppositories.
  • the active compounds are formulated into ointments, salves, gels, or creams as generally known in the art.
  • the antibodies may also be prepared in the form of suppositories (e.g., with conventional suppository bases such as cocoa butter and other glycerides) or retention enemas for rectal delivery.
  • suppositories e.g., with conventional suppository bases such as cocoa butter and other glycerides
  • retention enemas for rectal delivery.
  • the presently disclosed antibodies are prepared with carriers that will protect the compound against rapid elimination from the body, such as a controlled release formulation, including implants and microencapsulated delivery systems.
  • a controlled release formulation including implants and microencapsulated delivery systems.
  • Biodegradable, biocompatible polymers can be used, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid. Methods for preparation of such formulations will be apparent to those skilled in the art. The materials can also be obtained commercially from Alza Corporation and Nova Pharmaceuticals, Inc.
  • Liposomal suspensions containing the presently disclosed antibodies can also be used as pharmaceutically acceptable carriers. These can be prepared according to methods known to those skilled in the art, for example, as described in U.S. Patent No. 4,522,811 .
  • Dosage unit form refers to physically discrete units suited as unitary dosages for the subject to be treated; each unit containing a predetermined quantity of active compound calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier.
  • the specification for the dosage unit forms of the invention are dictated by and directly dependent on the unique characteristics of the active compound and the particular therapeutic effect to be achieved, and the limitations inherent in the art of compounding such an active compound for the treatment of individuals.
  • Toxicity and therapeutic efficacy of such compounds can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g ., for determining the LD 50 (the dose lethal to 50% of the population) and the ED 50 (the dose therapeutically effective in 50% of the population).
  • the dose ratio between toxic and therapeutic effects is the therapeutic index and it can be expressed as the ratio LD 50 /ED 50 .
  • Antibodies which exhibit large therapeutic indices are an embodiment of the invention.
  • the data obtained from the cell culture assays and animal studies can be used in formulating a range of dosage for use in humans.
  • the dosage of such compounds lies optionally within a range of circulating concentrations that include the ED 50 with little or no toxicity.
  • the dosage may vary within this range depending upon the dosage form employed and the route of administration utilized.
  • the therapeutically effective dose can be estimated initially from cell culture assays.
  • a dose may be formulated in animal models to achieve a circulating plasma concentration range that includes the IC 50 (i.e ., the concentration of the test antibody which achieves a half-maximal inhibition of symptoms) as determined in cell culture.
  • Levels in plasma may be measured, for example, by high performance liquid chromatography.
  • bioassays include DNA replication assays, transcription-based assays, GDF protein/receptor binding assays, creatine kinase assays, assays based on the differentiation of pre-adipocytes, assays based on glucose uptake in adipocytes, and immunological assays.
  • amino acids may be substituted for other amino acids in a protein structure without adversely affecting the activity of the protein, e.g., binding characteristics of an antibody. It is thus contemplated by the inventors that various changes may be made in the amino acid sequences of the presently disclosed antibodies, or DNA sequences encoding the antibodies, without appreciable loss of their biological utility or activity. Such changes may include deletions, insertions, truncations, substitutions, fusions, shuffling of motif sequences, and the like.
  • the hydropathic index of amino acids may be considered.
  • the importance of the hydropathic amino acid index in conferring interactive biological function on a protein is generally understood in the art ( Kyte and Doolittle (1982) J. Mol. Biol., 157: 105-132 ). It is accepted that the relative hydropathic character of the amino acid contributes to the secondary structure of the resultant protein, which in turn defines the interaction of the protein with other molecules, for example, enzymes, substrates, receptors, DNA, antibodies, antigens, and the like.
  • Each amino acid has been assigned a hydropathic index on the basis of its hydrophobicity and charge characteristics (Kyte and Doolittle, 1982); these are isoleucine (+4.5), valine (+4.2), leucine (+3.8), phenylalanine (+2.8), cysteine/cystine (+2.5), methionine (+1.9), alanine (+1.8), glycine (-0.4), threonine (-0.7), serine (-0.8), tryptophan (-0.9), tyrosine (-1.3), proline (-1.6), histidine (-3.2), glutamate (-3.5), glutamine (-3.5), aspartate (-3.5), asparagine (-3.5), lysine (-3.9), and arginine (-4.5).
  • substitution of amino acids whose hydropathic indices are within ⁇ 2 is an embodiment of the invention, those which are within ⁇ 1 are optional, and those within ⁇ 0.5 are also optional.
  • hydrophilicity values have been assigned to amino acid residues: arginine (+3.0), lysine (+3.0), aspartate (+3.0 ⁇ 1), glutamate (+3.0 ⁇ 1), serine (+0.3), asparagine (+0.2), glutamine (+0.2), glycine (0), threonine (-0.4), proline (-0.5 ⁇ 1), alanine (-0.5), histidine (-0.5), cysteine (-1.0), methionine (-1.3), valine (-1.5), leucine (-1.8), isoleucine (-1.8), tyrosine (-2.3), phenylalanine (-2.5), and tryptophan (-3.4).
  • substitution of amino acids whose hydrophilicity values are within ⁇ 2 is an embodiment of the invention, those within ⁇ 1 are optional, and those within ⁇ 0.5 are optional.
  • the modifications may be conservative such that the structure or biological function of the protein is not affected by the change.
  • conservative amino acid modifications are based on the relative similarity of the amino acid side-chain substituents, for example, their hydrophobicity, hydrophilicity, charge, size, and the like.
  • Exemplary conservative substitutions which take various of the foregoing characteristics into consideration are well known to those of skill in the art and include: arginine and lysine; glutamate and aspartate; serine and threonine; glutamine and asparagine; and valine, leucine, and isoleucine.
  • the amino acid sequence of the presently disclosed antibodies may be modified to have any number of conservative changes, so long as the binding of the antibody to its target antigen is not adversely affected. Such changes may be introduced inside or outside of the antigen binding portion of the antibody. For example, changes introduced inside of the antigen binding portion of the antibody may be designed to increase the affinity of the antibody for its target.
  • the antibodies can be glycosylated, pegylated, or linked to albumin or a nonproteinaceous polymer.
  • the presently disclosed antibodies may be linked to one of a variety of nonproteinaceous polymers, e.g., polyethylene glycol, polypropylene glycol, or polyoxyalkylenes, in the manner set forth in U.S. Patent Numbers 4,640,835 ; 4,496,689 ; 4,301,144 ; 4,670,417 ; 4,791,192 ; or 4,179,337 .
  • the antibodies are chemically modified by covalent conjugation to a polymer to increase their circulating half-life, for example. Certain polymers, and methods to attach them to peptides, are also shown in U.S. Pat. Nos. 4,766,106 ; 4,179,337 ; 4,495,285 ; and 4,609,546 .
  • the antibody may be modified to have an altered glycosylation pattern (i.e ., altered from the original or native glycosylation pattern).
  • altered means having one or more carbohydrate moieties deleted, and/or having one or more glycosylation sites added to the original antibody.
  • N-linked refers to the attachment of the carbohydrate moiety to the side chain of an asparagine residue.
  • the tripeptide sequences asparagine-X-serine and asparagine-X-threonine, where X is any amino acid except proline, are the recognition sequences for enzymatic attachment of the carbohydrate moiety to the asparagine side chain.
  • X is any amino acid except proline
  • O-linked glycosylation refers to the attachment of one of the sugars N-aceylgalactosamine, galactose, or xylose to a hydroxyamino acid, most commonly serine or threonine, although 5-hydroxyproline or 5-hydroxylysine may also be used.
  • glycosylation sites to the presently disclosed antibodies is conveniently accomplished by altering the amino acid sequence such that it contains one or more of the above-described tripeptide sequences (for N-linked glycosylation sites).
  • the alteration may also be made by the addition of, or substitution by, one or more serine or threonine residues to the sequence of the original antibodies (for O-linked glycosylation sites).
  • the antibody amino acid sequence is optionally altered through changes at the DNA level.
  • Another means of increasing the number of carbohydrate moieties on the antibodies is by chemical or enzymatic coupling of glycosides to the amino acid residues of the antibody. These procedures are advantageous in that they do not require production of the GDF peptide inhibitor in a host cell that has glycosylation capabilities for N- or O-linked glycosylation.
  • the sugars may be attached to (a) arginine and histidine, (b) free carboxyl groups, (c) free sulfhydryl groups such as those of cysteine, (d) free hydroxyl groups such as those of serine, threonine, or hydroxyproline, (e) aromatic residues such as those of phenylalanine, tyrosine, or tryptophan, or (f) the amide group of glutamine.
  • Removal of any carbohydrate moieties present on the antibodies may be accomplished chemically or enzymatically. Chemical deglycosylation requires exposure of the antibody to trifluoromethanesulfonic acid, or an equivalent compound. This treatment results in the cleavage of most or all sugars except the linking sugar (N-acetylglucosamine or N-acetylgalactosamine), while leaving the amino acid sequence intact.
  • the present invention includes these amino acid and nucleic acid sequences.
  • the present invention also include variants, homologues, and fragments of these nucleic and amino acid sequences.
  • the antibody may comprise a heavy chain variable region sequence that comprises SEQ ID NO:1, or a nucleic acid sequence that encodes SEQ ID NO:1 ( e.g ., SEQ ID NO:6).
  • the nucleic or amino acid sequence optionally comprises a sequence at least 70% to 79% identical to the nucleic or amino acid sequence of the presently disclosed variable heavy chain region, optionally at least 80% to 89% identical, optionally at least 90% to 95% identical, and optionally at least 96% to 100% identical.
  • the CDR region which determines the antigenic binding properties of the antibody, can tolerate less sequence variation than the other portions of the antibody not involved in antigen binding. Thus, these non-binding regions of the antibody may contain substantial variations without significantly altering the binding properties of the antibody.
  • affinity-increasing changes are typically determined empirically by altering the CDR region and testing the antibody. All such alterations, whether within the CDR or outside the CDR, are included in the scope of the present invention.
  • Relative sequence similarity or identity may be determined using the "Best Fit” or “Gap” programs of the Sequence Analysis Software PackageTM (Version 10; Genetics Computer Group, Inc., University of Wisconsin Biotechnology Center, Madison, WI). "Gap” utilizes the algorithm of Needleman and Wunsch (Needleman and Wunsch, 1970) to find the alignment of two sequences that maximizes the number of matches and minimizes the number of gaps. "BestFit” performs an optimal alignment of the best segment of similarity between two sequences. Optimal alignments are found by inserting gaps to maximize the number of matches using the local homology algorithm of Smith and Waterman (Smith and Waterman, 1981; Smith et al ., 1983).
  • the Sequence Analysis Software Package described above contains a number of other useful sequence analysis tools for identifying homologues of the presently disclosed nucleotide and amino acid sequences.
  • the "BLAST" program Altschul et al., 1990 searches for sequences similar to a query sequence (either peptide or nucleic acid) in a specified database (e.g ., sequence databases maintained at the National Center for Biotechnology Information (NCBI) in Bethesda, MD); "FastA” (Lipman and Pearson, 1985; see also Pearson and Lipman, 1988; Pearson et al ., 1990) performs a Pearson and Lipman search for similarity between a query sequence and a group of sequences of the same type (nucleic acid or protein); "TfastA” performs a Pearson and Lipman search for similarity between a protein query sequence and any group of nucleotide sequences (it translates the nucleotide sequences in all six reading frames before performing the comparison); "Fast
  • Conditioned media from a selected cell line expressing full-length human GDF-8 protein were acidified to pH 6.5 and applied to a 80 x 50 mm POROS HQ anion exchange column in tandem to a 80 x 50 mm POROS SP cation exchange column (PerSeptive Biosystems, Foster City, CA). The flow through was adjusted to pH 5.0 and applied to a 75 x 20 mm POROS SP cation exchange column (PerSeptive Biosystems) and eluted with a NaCl gradient.
  • Fractions containing the GDF-8 as indicated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), were pooled, acidified with trifluoroacetic acid (TFA) to pH 2-3, then brought up to 200 ml with 0.1% TFA to lower the viscosity. The pool was then applied to a 250 x 21.2 mm C 5 column (Phenomenex, Torrance, CA) preceded by a 60 x 21.2 mm guard column (Phenomenex) and eluted with a TFA/CH 3 CN gradient, to separate mature GDF-8 from GDF-8 propeptide.
  • TFA trifluoroacetic acid
  • the apparent molecular weight of purified GDF-8 propeptide was 38 kDa under both reducing and nonreducing conditions. This indicates that the GDF-8 propeptide by itself is monomeric.
  • the difference between the apparent molecular weight and the predicted molecular weight of GDF-8 propeptide, - 26 kDa, may reflect the addition of carbohydrate, since its amino acid sequence contains a potential N-linked glycosylation site (McPherron et al ., 1997, supra).
  • GDF-8 propeptide DNA and amino acid sequence are set forth in McPherron and Lee (1997) Proc. Natl. Acad. Sci. USA, 94: 12457-12461 .
  • GDF-8 knockout mice were immunized every two weeks with mature GDF-8 protein (purified as described in Example 1) mixed in Freunds complete adjuvant for the first two immunizations, and incomplete Freunds adjuvant thereafter. Throughout the immunization period, blood was sampled and tested for the presence of circulating antibodies. At week 9, an animal with circulating antibodies was selected, immunized for three consecutive days, and sacrificed. The spleen was removed and homogenized into cells.
  • the spleen cells were fused to a myeloma fusion partner (line P3-x63-Ag8.653) using 50% PEG 1500 by an established procedure ( Oi & Herzenberg (1980) Selected Methods in Cellular Immunology, W. J. Freeman Co., San Francisco, CA, p. 351 ).
  • the fused cells were plated into 96-well microtiter plates at a density of 2 x 10 5 cells/well. After 24 hours, the cells were subjected to HAT selection ( Littlefield (1964) Science, 145: 709 ) effectively killing any unfused and unproductively fused myeloma cells.
  • Mature GDF-8 protein was prepared from CHO cells as described above and coated on polystyrene (for solid phase assays) or biotinylated (for a solution based assay). Neutralizing assays were also employed where the ActRIIB receptor was coated on a polystyrene plate and biotin GDF-8 binding was inhibited by the addition of hybridoma supernatant. Results identified hybridomas expressing GDF-8 antibodies. These positive clones were cultured and expanded for further study. These cultures remained stable when expanded and cell lines were cloned by limiting dilution and cryopreserved.
  • a panel of synthetic peptides corresponding to portions of the GDF-8 protein sequence was produced.
  • Fig. 1 shows the GDF-8 synthetic peptides used in this study.
  • Even number peptides (N2-N14) were biotinylated on the primary amine.
  • the biotinylated peptides N2, N4, N6, N8, N10, N12, N14, and an irrelevant peptide DAE-10, were coated at 1 ⁇ g/ml for 2 hrs at room temperature on Reacti-BindTM Streptavidin coated polystyrene 96 well plates (Pierce, Rockford, IL, Cat. No.15124) following the manufacturer's protocol.
  • JA-16 or a unrelated monoclonal antibody control was added to the ELISA plate at 100, 10, and 1 nM (JA-16 only), and incubated for 30 min. After washing the plate, a secondary antibody (goat anti-murine IgG
  • GDF-8 and BMP-11 are 90% homologous at the amino acid level ( Fig. 3 ). Three of these changes are present within the N8 peptide. To compare the specificity of JA-16 towards GDF-8 and BMP-11, shorter peptides were designed G1 and B1 specific for GDF-8 and BMP-11, respectively. Differences between G1 and B1 are indicated with underlining.
  • G1 Asp-Phe- Gly-Leu-Asp-Ser-Asp-Glu-His-Ser- Thr -Glu-Ser-Arg-Cys (SEQ ID NO:10)
  • the peptides G1 and B1 were conjugated to BSA using a PIERCE conjugation kit (Cat. No. 77116ZZ) following manufacturer's protocol.
  • G1-BSA and B1-BSA were coated on 96 well flat-bottom assay plates (Costar, NY, Cat. No. 3590) at 1 ⁇ g/ml in 0.2 M sodium carbonate buffer overnight at 4°C. The plates were washed and blocked with PBS, 1 mg/ml BSA, 0.05 % Tween for 1 hour at room temperature.
  • JA-16 (5 nM) was serially diluted (1:2). The dilutions were added to the ELISA plate and incubated for 30 min at RT.
  • a secondary antibody (goat anti-murine IgG (H+L)-HRP, Calbiochem, Cat. No. 401215) was added at a 1:1000 dilution and incubated for 30 min at RT. Plates were washed four times, and TMB substrate was added (KPL, Cat. No. 50-76-04). Colorimetric measurements were done at 450 nm in a Molecular Devices microplate reader.
  • Fig. 4 shows that JA-16 binds to G1-BSA in a concentration dependent manner, but not to B1-BSA, even at the highest concentration.
  • G1-BSA was coated as described above, but this time, JA-16 at 5 nM was preincubated with either G1 peptide or B1 peptide, GDF-8, or BMP-11 at various concentrations. The result is shown in Fig. 5 .
  • the BMP-11 specific peptide B1 does not inhibit binding of JA-16 to G1-BSA, but G1 does.
  • the IC 50 for GDF-8 is 0.8 ⁇ g/ml, while BMP-11's IC 50 is 3.8 ⁇ g/ml demonstrating that JA-16 recognizes GDF-8 with a 5-fold higher affinity than BMP-11.
  • the array was defined on the membrane by coupling a ⁇ -alanine spacer and peptides were synthesized using standard DIC (diisopropylcarbodiimide)/HOBt (hydroxybenzotriazole) coupling chemistry as described previously ( Molina et al. (1996) Peptide Research, 9: 151-155 ; Frank et al. (1992) Tetrahedron, 48: 9217-9232 ).
  • Activated amino acids were spotted using an Abimed ASP 222 robot. Washing and deprotection steps were done manually and the peptides were N-terminally acetylated after the final synthesis cycle. Following peptide synthesis, the membrane was washed in methanol for 10 minutes and in blocker (TBST (Tris buffered saline with 0.1 % (v/v) Tween 20) + 1% (w/v) casein) for 10 minutes. The membrane was then incubated with 2.5 ⁇ g/ml JA-16 in blocker for 1 hour with gentle shaking. After washing with blocker 3 times for 10 minutes, the membrane was incubated with HRP-labeled secondary antibody (0.25 ⁇ g/ml in blocker) for 30 minutes.
  • TST Tris buffered saline with 0.1 % (v/v) Tween 20
  • HRP-labeled secondary antibody 0.25 ⁇ g/ml in blocker
  • JA-16 bound to the first 4 peptides of the array (SEQ ID NOS:17-20), which corresponds to 18 residues on the N-terminus of GDF-8.
  • deletion and substitution analyses of the peptide Gly-Leu-Asp-Ser-Asp-Glu-His-Ser-Thr-Glu-Ser-Arg-Ser were performed using spot synthesis.
  • substitution analysis each residue of this peptide was individually replaced with each of the 20 natural amino acids except cysteine, generating SEQ ID NOS: 3, 18, 66-104, 106-113, and 115-128. Synthesis and binding assays were performed as described above. The results are shown in Fig. 7 .
  • JA-16 was tested for its ability to inhibit mature GDF-8 protein binding to the ActRIIB receptor.
  • Recombinant ActRIIB.Fc chimera R&D Systems, Minneapolis, MN, Cat. No. 339-RB/CF
  • ActRIIB.Fc chimera R&D Systems, Minneapolis, MN, Cat. No. 339-RB/CF
  • 96 well flat-bottom assay plates Costar, Cat. No. 3590
  • JA-16 has a very weak in vitro neutralizing activity of around 1 ⁇ M. This in vitro data suggests that JA-16 is unlikely to be a very good neutralizer of active GDF-8, particularly under less controlled in vivo conditions.
  • a reporter gene assay was performed to assess the biological activity of active GDF-8 protein in vitro.
  • the assay uses a reporter vector, pGL3(CAGA) 12 , coupled to luciferase.
  • the CAGA sequence was previously reported to be a TGF- ⁇ -responsive sequence within the promoter of the TGF- ⁇ -induced gene, PAI-1.
  • the reporter vector containing 12 CAGA boxes was made using the basic reporter plasmid, pGL3 (Promega Corporation, Madison, WI, Cat. No. E1751).
  • the TATA box and transcription initiation site from the adenovirus major late promoter (-35/+10) was inserted between the BgIII and HindIII sites.
  • Oligonucleotides containing twelve repeats of the CAGA boxes AGCCAGACA were annealed and cloned into the XhoI site.
  • the human rhabdomyosarcoma cell line, A204 was transiently transfected with pGL3(CAGA) 12 using FuGENE 6 transfection reagent (Roche Diagnostics, Indianapolis, MN Cat. No.
  • GDF-8 maximally activated the reporter construct 10-fold, with an ED 50 of 10 ng/ml GDF-8.
  • BMP-11 which is 90% identical to GDF-8 at the amino acid level ( Gamer et al. (1999) Dev. Biol., 208(1): 222-32 ; Nakashima et al. (1999) Mech. Dev., 80(2): 185-9 ), and activin elicited a similar biological response.
  • JA-16's neutralizing activity was determined by preincubating JA-16 with mature GDF-8 protein for 30 min prior to addition to the A204 cells.
  • An irrelevant antibody monoclonal control
  • a human GDF-8 antibody derived from scFv phagemid library using phage display technology (Myo-19) were also tested.
  • Fig. 10 shows that, in this assay as well, JA-16 is weakly neutralizing with an IC 50 of around 1 ⁇ M, while the Myo-19 IC 50 is around 100 nM. Based on this in vitro data, one would have expected the Myo-19 antibody to be a better neutralizer of active GDF-8 protein than JA-16 in vivo, which is not the case, as shown herein.
  • CHO cells expressing GDF-8 were radiolabeled with 35 S-methionine and 35 S-cysteine.
  • 100 ⁇ l of conditioned medium from these cells containing GDF-8 latent complex was incubated with 1 mg/ml JA-16 for 1 hour at 4°C.
  • Protein A Sepharose was added to the mixture, which was then incubated overnight at 4°C.
  • the immunoprecipitate was collected, washed three times with a PBS/Triton-X100 buffer, resuspended in reducing sample buffer and analyzed by SDS-PAGE. The gel was fixed overnight, enhanced with autoradiography enhancer solution, dried and the autoradiogram was developed.
  • Figure 18 , lane 2 shows that JA-16 can immunoprecipitate the GDF-8 latent complex and unprocessed GDF-8.
  • CHO cells expressing follistatin were radiolabeled with 35 S-methionine and 35 S-cysteine.
  • 100 ⁇ l of conditioned medium containing radiolabeled follistatin was mixed with mature GDF-8 to form a complex of GDF-8 with follistatin.
  • the mixture was incubated with 1 mg/ml JA-16 for 1 hour at 4°C.
  • Protein A Sepharose was added to the mixture, which was then incubated overnight at 4°C.
  • the immunoprecipitate was collected and analyzed as described above.
  • conditioned media from CHO cells containing radiolabeled GDF-8 latent complex was acid activated to dissociate the GDF-8 propeptide and mature GDF-8 (see van Waarde et al. (1997) Analytical Biochemistry, 247, 45-51 ). This material was then incubated with JA-16 for 1 hour at 4°C. The remainder of the protocol was performed as described above.
  • mice were weighed and evenly distributed with respect to body weight into groups of seven or eight.
  • JA-16 in PBS or an isotype matched antibody to snake venom (control) was injected into the mice intraperitoneally at 50 mg/kg twice weekly. The treatment continued for four weeks. Animals were assessed for gain in lean body mass by subjecting them to dexascan analysis before and after the treatment period. Muscle mass was assessed by dissecting and weighing the gastrocnemius and quadriceps. The peri-uterine fat pad was also removed and weighed. The results of this study indicated that JA-16 significantly inhibits GDF-8 activity in vivo resulting in increased muscle mass ( Fig. 11 ).
  • mice were loaded at the beginning of the study with 60 mg/kg intraperitoneally and 10 mg/kg intravenously.
  • the mice in this study were male C57BL mice that were either wild type at the agouti locus (a) or carried the lethal yellow mutation (Ay) at that locus.
  • the Ay mutation causes adult onset obesity and diabetes, which allowed us to determine the effect of JA-16 on muscle, excess fat, and blood glucose in a diabetic background.
  • Total body mass was measured weekly ( Fig. 12 ). Muscle mass was assessed by dissecting and weighing the gastrocnemius and quadriceps ( Fig. 13 ).
  • the in vivo activity of JA-16 was also compared to the in vivo activity of another GDF-8 antibody, Myo-19.
  • C57B6/scid mice we injected intraperitoneally for five weeks with vehicle control or with 60 mg/kg loading dose plus 60 mg/kg per week of JA-16 or Myo-19.
  • Total body mass was measured weekly and muscle mass was assessed by dissecting and weighing the gastrocnemius and quadriceps ( Fig. 17 ). While five weeks of treatment with JA-16 led to an increase in muscle mass, treatment with Myo-19 did not effect muscle mass.
  • Myo-19 treatment was extended to 10 and to 15 weeks, and no increase in body mass or muscle mass was seen for these time points.
  • Example 9 JA-16 Increases Muscle Strength
  • mice In humans, muscle size and strength decreases by approximately 1 % per year starting in the third decade of life. For many aged people, the loss in muscle mass is significantly debilitating. This condition is known as sarcopenia, or age related loss of muscle.
  • sarcopenia or age related loss of muscle.
  • aged mice (19 months of age at the beginning of the study and 21 months of age at the end of the study) were treated with JA-16 for 8 weeks at 60 mg/kg once a week.
  • young mice (2 month of age at the beginning of the study and 4 months of age at the end of the study) were treated with the same dose of JA-16. At the end of the study, both groups of mice had greater muscle mass than the vehicle treated controls as seen, for example, from the quadriceps mass comparison ( Fig.19A ).
  • Example 10 JA-16 Increases Muscle Mass and Strength in Dystrophic Muscle
  • DMD Duchenne's muscular dystrophy
  • mice Four week old male mdx mice were treated with weekly intraperitoneal injections of JA-16 (60 mg/kg), and vehicle alone (control group) for 3 months.
  • JA-16 60 mg/kg
  • vehicle alone control group
  • EDL extensor digitorum longus
  • Figure 20A EDL muscles from the treated group of animals weighed significantly more than controls.
  • the relative increase in muscle mass was greater than the increase in body weight as shown in Figure 20B .
  • other muscle groups including the gastrocnemius, tibialis anterior and quadriceps were found to have similar increases in weight.
  • GDF-8 knockout mice were assessed for altered bone mass and microarchitecture. An initial assessment of adult mice showed that bone density in the spine of the KO mice was nearly two-fold higher than that of their wild-type littermates. This increase far exceeded what might have been expected to be solely due to the increased muscle mass in the GDF-8 KO mice.
  • High resolution microtomographic imaging ( ⁇ CT40, Scanco Medical, Switzerland) was used to assess the trabecular bone volume fraction and microarchitecture in the 5th lumbar vertebrae and distal femora and cortical bone geometry at the femoral mid-diaphysis of adult GDF-8 wildtype (WT) and KO mice. Specimens were taken from 9-10 month old GDF-8 male and female KO and littermate controls (four mice of each genotype and sex). The entire vertebral body and femur were scanned using microcomputed tomography at 12 ⁇ m resolution.
  • Regions of interest encompassing the trabecular bone of the vertebral body or the trabecular bone of the distal femoral metaphysis (i.e ., secondary spongiosa) were identified using a semi-automated contouring algorithm. The following parameters were computed using direct 3D assessments: bone volume fraction (%), trabecular thickness ( ⁇ m), separation ( ⁇ m) and number (1/mm). In addition, the connectivity density, an indicator of how well the trabecular network is connected, was assessed as well as cortical bone parameters at the middiaphyseal region in the femur, including total area, bone area, and cortical thickness.
  • Both male and female KO mice had dramatically increased trabecular bone density in the vertebral body compared to WT littermates (n 4, +93% and +70%, respectively, p ⁇ 0.0001).
  • Inhibitors of GDF-8 are useful for treatments directed at increased muscle mass, and also for prevention and treatment of osteoporosis.
  • inhibition of GDF-8 may be useful in other instances where a bone anabolic effect is desired, such as augmentation of bone healing (i.e., fracture repair, spine fusion, etc.).
  • the anti-GDF-8 antibodies of the invention are used to treat a subject at disease onset or having an established muscle or bone degenerative disease.
  • Efficacy of anti-GDF-8 antibodies for treatment of bone disorders is confirmed using well established models of osteoporosis.
  • ovariectomized mice have been used to test the efficacy of new osteoporosis drug treatments ( Alexander et al. (2001) J. Bone Min. Res. 16: 1665-1673 ; and Anderson et al. (2001) J. Endocrinol. 170:529-537 ). Similar to humans, these rodents exhibit a rapid loss of bone following ovariectomy, especially in cancellous bone.
  • Outcome assessments are based on bone mineral density, biochemical markers of bone turnover in serum and urine, bone strength, and histology/histomorphometry.
  • mice normal and/or immune compromised female mice are ovariectomized at 12-16 weeks of age and allowed to lose bone for four to six weeks. Following this bone loss period, treatment with an anti-GDF-8 antibody such as JA-16 (IP injection) or vehicle is conducted for one to six months.
  • the treatment protocol could vary, with testing of different doses and treatment regimens (e.g., daily, weekly, or bi-weekly injections), It is anticipated that untreated ovariectomized mice (or rats) would lose approximately 10-30% of bone density relative to intact (i.e., non-ovariectomized), age-matched mice.
  • mice treated with the anti-GDF-8 antibody would have 10 to 50% greater bone mass and bone density than those mice receiving placebo, and moreover that this increase in bone density would be associated with increased bone strength, particularly in regions with a greater proportion of cancellous bone compared to cortical bone.
  • anti-GDF-8 antibody such as JA-16 is effective in preventing the decline in bone mass, microarchitecture and strength associated with estrogen deficiency.
  • the study has a similar design to the one described above, except that treatment with anti-GDF-8 antibody would be initiated immediately after ovariectomy, rather than after the bone loss period. It is anticipated that mice treated with the antibody would lose significantly less bone mass following ovariectomy than mice treated with vehicle.
  • the inhibitory antibodies against GDF-8 are also used to prevent and/or to reduce severity and/or the symptoms of the disease. It is anticipated that the anti-GDF-8 antibodies would be administered as a subcutaneous injection as frequently as once per day and as infrequently as once per month. Treatment duration could range between one month and several years.
  • Treatment groups include a placebo group and one to three groups receiving antibody (different doses). Individuals are followed prospectively for one to three years to assess changes in biochemical markers of bone turnover, changes in bone mineral density, and the occurrence of fragility fractures. It is anticipated that individuals receiving treatment would exhibit an increase in bone mineral density in the proximal femur and lumbar spine of 2-30% relative to baseline, and would have a decreased incidence of fragility fractures. It is anticipated that biochemical markers of bone formation would increase.
  • the antibodies are administered as the sole active compound or in combination with another compound or composition.
  • the dosage When administered as the sole active compound or in combination with another compound or composition, the dosage may be between approximately 1 ⁇ g/kg and 20 mg/kg, depending on the severity of the symptoms and the progression of the disease.
  • the appropriate effective dose is selected by a treating clinician from the following ranges: 1 ⁇ g/kg and 20 mg/kg, 1 ⁇ g/kg and 10 mg/kg, 1 ⁇ g/kg and 1 mg/kg, 10 ⁇ g/kg and 1 mg/kg, 10 ⁇ g/kg and 100 ⁇ g/kg, 100 ⁇ g and 1 mg/kg, and 500 ⁇ g/kg and 1 mg/kg.
  • Exemplary treatment regimens and outcomes are summarized in Table 4.
  • Inhibitors of GDF-8 are useful for treatment of metabolic disorders such as type 2 diabetes, impaired glucose tolerance, metabolic syndrome (e.g., syndrome X), insulin resistance induced by trauma (e.g., burns), and adipose tissue disorders (e.g., obesity).
  • the anti-GDF-8 antibodies of the invention are used to treat a subject at disease onset or having an established metabolic disease.
  • Efficacy of anti-GDF-8 antibodies for treatment of metabolic disorders is confirmed using well established murine models of obesity, insulin resistance and type 2 diabetes, including ob/ob, db/db, and strains carrying the lethal yellow mutation.
  • Insulin resistance can also be induced by high fat or high caloric feeding of certain strains of mice including, C57BL/6J. Similar to humans, these rodents develop insulin resistance, hyperinsuliemia, dyslipidemia, and deterioration of glucose homeostasis resulting in hyperglycemia.
  • Outcome assessments are based on serum measurements of glucose, insulin, and lipids. Improved insulin sensitivity can be determined by insulin tolerance tests and glucose tolerance tests.
  • More sensitive techniques would include the use of euglycemic-hyperinsulinemic clamps for assessing improvements is glycemic control and insulin sensitivity.
  • the clamp techniques would allow a quantitative assessment of the role of the major glucose disposing tissues (e.g., muscle, adipose, and liver) in improved glycemic control.
  • treatment with an anti-GDF-8 antibody such as JA-16 (IP injection) or vehicle is conducted for one week to six months.
  • the treatment protocol could vary, with testing of different doses and treatment regimens (e.g., daily, weekly, or bi-weekly injections). It is anticipated that mice treated with the anti-GDF-8 antibody would have greater glucose uptake, increased glycolysis and glycogen synthesis, lower free fatty acids and triglycerides in the serum as compared to mice receiving placebo treatment.
  • the inhibitory antibodies against GDF-8 are also used to prevent and/or to reduce severity and/or the symptoms of the disease. It is anticipated that the anti-GDF-8 antibodies would be administered as a subcutaneous injection as frequently as once per day and as infrequently as once per month. Treatment duration could range between one month and several years.
  • Treatment groups include a placebo group and one to three groups receiving antibody (different doses). Individuals are followed prospectively for one month to three years to assess changes in glucose metabolism. It is anticipated that individuals receiving treatment would exhibit an improvement.
  • the antibodies are administered as the sole active compound or in combination with another compound or composition.
  • the dosage When administered as the sole active compound or in combination with another compound or composition, the dosage may be between approximately 1 ⁇ g/kg and 20 mg/kg, depending on the severity of the symptoms and the progression of the disease.
  • the appropriate effective dose is selected by a treating clinician from the following ranges: 1 ⁇ g/kg and 20 mg/kg, 1 ⁇ g/kg and 10 mg/kg, 1 ⁇ g/kg and 1 mg/kg, 10 ⁇ g/kg and 1 mg/kg, 10 ⁇ g/kg and 100 ⁇ g/kg, 100 ⁇ g and 1 mg/kg, and 500 ⁇ g/kg and 1 mg/kg.
  • Exemplary treatment regimens and outcomes are summarized in Table 5.

Landscapes

  • Health & Medical Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • General Health & Medical Sciences (AREA)
  • Medicinal Chemistry (AREA)
  • Animal Behavior & Ethology (AREA)
  • Veterinary Medicine (AREA)
  • General Chemical & Material Sciences (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Public Health (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Engineering & Computer Science (AREA)
  • Physical Education & Sports Medicine (AREA)
  • Diabetes (AREA)
  • Orthopedic Medicine & Surgery (AREA)
  • Biophysics (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Molecular Biology (AREA)
  • Genetics & Genomics (AREA)
  • Biochemistry (AREA)
  • Obesity (AREA)
  • Hematology (AREA)
  • Neurology (AREA)
  • Rheumatology (AREA)
  • Immunology (AREA)
  • Zoology (AREA)
  • Toxicology (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Endocrinology (AREA)
  • Child & Adolescent Psychology (AREA)
  • Biomedical Technology (AREA)
  • Nutrition Science (AREA)
  • Neurosurgery (AREA)
  • Emergency Medicine (AREA)
  • Pulmonology (AREA)
  • Peptides Or Proteins (AREA)
  • Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)
EP02783984.4A 2001-09-26 2002-09-26 Antibody inhibitors of gdf-8 and uses thereof Expired - Lifetime EP1438068B1 (en)

Priority Applications (1)

Application Number Priority Date Filing Date Title
EP10183355A EP2316851A1 (en) 2001-09-26 2002-09-26 Antibody inhibitors of GDF-8 and uses thereof

Applications Claiming Priority (3)

Application Number Priority Date Filing Date Title
US32452801P 2001-09-26 2001-09-26
US324528P 2001-09-26
PCT/US2002/030452 WO2003027248A2 (en) 2001-09-26 2002-09-26 Antibody inhibitors of gdf-8 and uses thereof

Related Child Applications (1)

Application Number Title Priority Date Filing Date
EP10183355A Division-Into EP2316851A1 (en) 2001-09-26 2002-09-26 Antibody inhibitors of GDF-8 and uses thereof

Publications (3)

Publication Number Publication Date
EP1438068A2 EP1438068A2 (en) 2004-07-21
EP1438068A4 EP1438068A4 (en) 2005-11-16
EP1438068B1 true EP1438068B1 (en) 2014-05-21

Family

ID=23263987

Family Applications (2)

Application Number Title Priority Date Filing Date
EP02783984.4A Expired - Lifetime EP1438068B1 (en) 2001-09-26 2002-09-26 Antibody inhibitors of gdf-8 and uses thereof
EP10183355A Withdrawn EP2316851A1 (en) 2001-09-26 2002-09-26 Antibody inhibitors of GDF-8 and uses thereof

Family Applications After (1)

Application Number Title Priority Date Filing Date
EP10183355A Withdrawn EP2316851A1 (en) 2001-09-26 2002-09-26 Antibody inhibitors of GDF-8 and uses thereof

Country Status (16)

Country Link
US (5) US7320789B2 (zh)
EP (2) EP1438068B1 (zh)
JP (2) JP4452077B2 (zh)
AU (1) AU2002347773B2 (zh)
BR (1) BR0212809A (zh)
CA (1) CA2469230C (zh)
DK (1) DK1438068T3 (zh)
ES (1) ES2481165T3 (zh)
HK (1) HK1064284A1 (zh)
MX (1) MXPA04002834A (zh)
NO (1) NO336528B1 (zh)
NZ (1) NZ532034A (zh)
PL (1) PL374221A1 (zh)
PT (1) PT1438068E (zh)
WO (1) WO2003027248A2 (zh)
ZA (2) ZA200402265B (zh)

Families Citing this family (101)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
TW200526779A (en) 2001-02-08 2005-08-16 Wyeth Corp Modified and stabilized GDF propeptides and uses thereof
US7320789B2 (en) 2001-09-26 2008-01-22 Wyeth Antibody inhibitors of GDF-8 and uses thereof
MXPA04008150A (es) 2002-02-21 2005-06-17 Wyeth Corp Gasp1: una proteina que contiene dominio de folistatina.
KR20040096592A (ko) * 2002-02-21 2004-11-16 와이어쓰 폴리스타틴 도메인을 포함하는 단백질
US20040138118A1 (en) * 2002-09-16 2004-07-15 Neil Wolfman Metalloprotease activation of myostatin, and methods of modulating myostatin activity
US7261893B2 (en) 2002-10-22 2007-08-28 Wyeth Neutralizing antibodies against GDF-8 and uses therefor
US20040223966A1 (en) * 2002-10-25 2004-11-11 Wolfman Neil M. ActRIIB fusion polypeptides and uses therefor
US7511012B2 (en) 2002-12-20 2009-03-31 Amgen Inc. Myostatin binding agents
CA2526669A1 (en) * 2003-06-02 2004-12-16 Wyeth Use of myostatin (gdf8) inhibitors in conjunction with corticosteroids for treating neuromuscular disorders
NZ547593A (en) 2003-12-31 2009-09-25 Schering Plough Ltd Neutralizing epitope-based growth enhancing vaccine
EP3006039B1 (en) 2004-03-02 2021-01-06 Acceleron Pharma Inc. Alk7 polypeptides for use in promoting fat loss
US20070178095A1 (en) * 2004-03-23 2007-08-02 Eli Lilly And Company Anti-myostatin antibodies
EP2332977B1 (en) * 2004-07-23 2015-11-25 Acceleron Pharma Inc. ActRII receptor polypeptides
WO2006020884A2 (en) * 2004-08-12 2006-02-23 Wyeth Combination therapy for diabetes, obesity, and cardiovascular diseases using gdf-8 inhibitors
US7300773B2 (en) 2004-08-27 2007-11-27 Wyeth Research Ireland Limited Production of TNFR-Ig
US7335491B2 (en) 2004-08-27 2008-02-26 Wyeth Research Ireland Limited Production of anti-abeta
RU2451082C2 (ru) * 2004-08-27 2012-05-20 Вайет Рисёрч Айрлэнд Лимитед Производство полипептидов
TWI384069B (zh) * 2004-08-27 2013-02-01 Pfizer Ireland Pharmaceuticals 多胜肽之製法
AU2005323087A1 (en) 2004-12-30 2006-07-13 Schering-Plough Pty. Limited Neutralizing epitope-based growth enhancing vaccine
NZ538097A (en) * 2005-02-07 2006-07-28 Ovita Ltd Method and compositions for improving wound healing
US20060240488A1 (en) * 2005-03-23 2006-10-26 Nowak John A Detection of an immune response to GDF-8 modulating agents
MX2007011400A (es) * 2005-03-23 2007-10-11 Wyeth Corp Deteccion de agentes moduladores de crecimiento y la diferenciacion del factor 8 (gdf 8).
TW200724548A (en) * 2005-04-25 2007-07-01 Pfizer Antibodies to myostatin
CA2538208A1 (en) * 2005-05-04 2006-11-04 Universite Laval Modulation of myostatin and use thereof in cell transplantation-based treatment of muscle disease
EP2407486B1 (en) * 2005-08-19 2017-11-22 Wyeth LLC Antagonist antibodies against GDF-8 and uses in treatment of ALS and other GDF-8-associated disorders
CA2624976A1 (en) * 2005-10-06 2007-04-19 Eli Lilly And Company Anti-myostatin antibodies
UA92504C2 (en) * 2005-10-12 2010-11-10 Эли Лилли Энд Компани Anti-myostatin monoclonal antibody
KR20190006086A (ko) 2005-11-23 2019-01-16 악셀레론 파마 인코포레이티드 액티빈-ａｃｔｒⅱａ 길항제 및 골 성장을 촉진하기 위한 이들의 용도
US8128933B2 (en) 2005-11-23 2012-03-06 Acceleron Pharma, Inc. Method of promoting bone growth by an anti-activin B antibody
EP1968621A2 (en) * 2005-12-06 2008-09-17 Amgen Inc. Uses of myostatin antagonists
EP4001409A1 (en) 2006-03-31 2022-05-25 Chugai Seiyaku Kabushiki Kaisha Methods for controlling blood pharmacokinetics of antibodies
JP5058261B2 (ja) * 2006-09-05 2012-10-24 イーライ リリー アンド カンパニー 抗ミオスタチン抗体
US8895016B2 (en) 2006-12-18 2014-11-25 Acceleron Pharma, Inc. Antagonists of activin-actriia and uses for increasing red blood cell levels
US20100028332A1 (en) * 2006-12-18 2010-02-04 Acceleron Pharma Inc. Antagonists of actriib and uses for increasing red blood cell levels
EP2446896A1 (en) * 2006-12-18 2012-05-02 Acceleron Pharma, Inc. Activin-ActRII Antagonists for use in Increasing Red Blood Cells, increasing reticulocyte levels, or promoting erythropoiesis
RU2473362C2 (ru) * 2007-02-01 2013-01-27 Акселерон Фарма Инк. АНТАГОНИСТЫ АКТИВИНА-ActRIIa-Fc И ИХ ПРИМЕНЕНИЕ ДЛЯ ЛЕЧЕНИЯ ИЛИ ПРОФИЛАКТИКИ РАКА МОЛОЧНОЙ ЖЕЛЕЗЫ
TWI548647B (zh) 2007-02-02 2016-09-11 艾瑟勒朗法瑪公司 衍生自ＡｃｔＲＩＩＢ的變體與其用途
EA025371B1 (ru) * 2007-02-09 2016-12-30 Акселерон Фарма Инк. АНТАГОНИСТЫ АКТИВИНА-ActRIIa И ПРИМЕНЕНИЕ ДЛЯ СТИМУЛЯЦИИ РОСТА КОСТИ У БОЛЬНЫХ РАКОМ
BRPI0811317A2 (pt) 2007-08-03 2015-01-27 Summit Corp Plc Combinação, embalagem farmcêutica, kit ou mbalagem par apaciente, agente auxiliar, compsoto, usos de um agente auxiliar e de um composto, e, processo para a produção de uma combinação
GB0715087D0 (en) 2007-08-03 2007-09-12 Summit Corp Plc Drug combinations for the treatment of duchenne muscular dystrophy
EP3243524A1 (en) 2007-09-18 2017-11-15 Acceleron Pharma Inc. Activin-actriia antagonists and uses for decreasing or inhibiting fsh secretion
DK2202245T3 (en) 2007-09-26 2016-11-21 Chugai Pharmaceutical Co Ltd A method of modifying an antibody isoelectric point VIA amino acid substitution in CDR
PE20091163A1 (es) * 2007-11-01 2009-08-09 Wyeth Corp Anticuerpos para gdf8
EP3521311A1 (en) 2008-04-11 2019-08-07 Chugai Seiyaku Kabushiki Kaisha Antigen-binding molecule capable of binding to two or more antigen molecules repeatedly
EP3363453A1 (en) * 2008-06-26 2018-08-22 Acceleron Pharma Inc. Soluble actriia as activin-actriia antagonist for use in treating anemia or bone-related disorders
US8216997B2 (en) 2008-08-14 2012-07-10 Acceleron Pharma, Inc. Methods for increasing red blood cell levels and treating anemia using a combination of GDF traps and erythropoietin receptor activators
LT3494986T (lt) 2008-08-14 2020-07-10 Acceleron Pharma Inc. Gdf gaudyklės
AR074777A1 (es) * 2008-12-19 2011-02-09 Glaxo Group Ltd Proteinas de union a antigeno
WO2010083034A1 (en) * 2009-01-13 2010-07-22 Acceleron Pharma Inc. Methods for increasing adiponectin
US8178488B2 (en) 2009-06-08 2012-05-15 Acceleron Pharma, Inc. Methods for increasing thermogenic adipocytes
KR20190090049A (ko) 2009-06-12 2019-07-31 악셀레론 파마 인코포레이티드 절두된 ＡｃｔＲＩＩＢ-ＦＣ 융합 단백질
US10704096B2 (en) 2009-07-07 2020-07-07 University Of Southern California Biomarkers for the early detection of autoimmune diseases
IN2012DN02766A (zh) * 2009-09-09 2015-09-18 Acceleron Pharma Inc
JP5836961B2 (ja) * 2009-11-03 2015-12-24 アクセルロン ファーマ， インコーポレイテッド 脂肪肝疾患を処置するための方法
ES2658292T3 (es) 2009-11-17 2018-03-09 Acceleron Pharma, Inc. Proteínas ActRIIB y variantes y usos de las mismas con respecto a la inducción de la utrofina para el tratamiento de la distrofia muscular
JO3340B1 (ar) 2010-05-26 2019-03-13 Regeneron Pharma مضادات حيوية لـعامل تمايز النمو 8 البشري
UY33421A (es) * 2010-06-03 2011-12-30 Glaxo Wellcome House Proteinas de union al antígeno humanizados
CN105440134A (zh) 2010-08-16 2016-03-30 安姆根公司 结合肌肉生长抑制素的抗体、组合物和方法
US20120121576A1 (en) 2010-11-08 2012-05-17 Jasbir Seehra Actriia binding agents and uses thereof
CA2819356C (en) 2010-11-30 2023-01-24 Chugai Seiyaku Kabushiki Kaisha Antigen-binding molecule capable of binding to plurality of antigen molecules repeatedly
LT2837680T (lt) 2011-07-01 2020-07-10 Amgen Inc. Žinduolių ląstelių kultūra
DK2780368T3 (en) 2011-11-14 2018-02-05 Regeneron Pharma COMPOSITIONS AND PROCEDURES FOR INCREASING MUSCLE MASS AND MUSCLE STRENGTH BY SPECIFIC ANTAGONIZATION OF GDF8 AND / OR ACTIVIN A
MX364738B (es) 2012-06-15 2019-05-03 Pfizer Anticuerpos antagonistas mejorados contra factor-8 de crecimiento y diferenciacion y usos de los mismos.
CA2880649C (en) 2012-08-01 2023-03-14 Elizabeth MCNALLY Mitigating tissue damage and fibrosis via latent transforming growth factor beta binding protein (ltbp4)
KR20230110836A (ko) 2012-08-24 2023-07-25 추가이 세이야쿠 가부시키가이샤 ＦｃγＲＩＩｂ 특이적 Ｆｃ영역 개변체
US11236168B2 (en) 2012-08-24 2022-02-01 Chugai Seiyaku Kabushiki Kaisha Mouse FcγammaRII-specific Fc antibody
CN104768969B (zh) 2012-09-13 2021-04-16 百时美施贵宝公司 结合至肌生成抑制素的基于纤连蛋白的支架结构域蛋白
EA201590693A1 (ru) 2012-10-05 2015-08-31 Ригель Фармасьютикалс, Инк. Ингибиторы gdf-8
NZ747350A (en) 2012-10-24 2020-07-31 Celgene Corp Methods for treating anemia
CA2890217C (en) 2012-11-02 2021-07-20 Yifu FANG Activin-actrii antagonists and uses for treating bone and other disorders
EP2982689B1 (en) 2013-04-02 2020-08-26 Chugai Seiyaku Kabushiki Kaisha Fc region variant
CA2911514A1 (en) * 2013-05-06 2014-11-13 Scholar Rock, Inc. Compositions and methods for growth factor modulation
TWI655207B (zh) 2013-07-30 2019-04-01 再生元醫藥公司 抗活化素ａ之抗體及其用途
US10233170B2 (en) * 2014-04-08 2019-03-19 Rigel Pharmaceuticals, Inc. 2,3-disubstituted pyridine compounds as TGF-beta inhibitors and methods of use
AU2015274277B2 (en) 2014-06-13 2021-03-18 Acceleron Pharma, Inc. Methods and compositions for treating ulcers
MA41052A (fr) 2014-10-09 2017-08-15 Celgene Corp Traitement d'une maladie cardiovasculaire à l'aide de pièges de ligands d'actrii
CA3005158A1 (en) 2014-11-06 2016-05-12 Scholar Rock, Inc. Anti-pro/latent-myostatin antibodies and uses thereof
EP3221318B1 (en) 2014-11-21 2021-08-25 Rigel Pharmaceuticals, Inc. Fused imidazole derivatives as tgf-beta inhibitors
DK3227675T3 (da) 2014-12-03 2023-05-30 Celgene Corp Activin-actrii-antagonister og anvendelser til behandling af myelodysplastisk syndrom
NZ730607A (en) 2014-12-19 2022-07-01 Chugai Pharmaceutical Co Ltd Anti-myostatin antibodies, polypeptides containing variant fc regions, and methods of use
ES2899894T3 (es) 2014-12-19 2022-03-15 Chugai Pharmaceutical Co Ltd Anticuerpos anti-C5 y métodos de uso
EA201791754A1 (ru) 2015-02-05 2019-01-31 Чугаи Сейяку Кабусики Кайся АНТИТЕЛА, СОДЕРЖАЩИЕ ЗАВИСЯЩИЙ ОТ КОНЦЕНТРАЦИИ ИОНОВ АНТИГЕНСВЯЗЫВАЮЩИЙ ДОМЕН, ВАРИАНТЫ Fc-ОБЛАСТИ, IL-8-СВЯЗЫВАЮЩИЕ АНТИТЕЛА И ИХ ПРИМЕНЕНИЯ
WO2016128523A1 (en) 2015-02-12 2016-08-18 INSERM (Institut National de la Santé et de la Recherche Médicale) Methods for predicting the responsiveness of a patient affected with malignant hematological disease to chemotherapy treatment and methods of treatment of such disease
ES2928084T3 (es) 2015-02-20 2022-11-15 Rigel Pharmaceuticals Inc Inhibidores de GDF-8
EP3265454B1 (en) 2015-03-02 2020-02-26 Rigel Pharmaceuticals, Inc. Tgf-beta inhibitors
EP3283479B1 (en) 2015-04-01 2022-12-14 Rigel Pharmaceuticals, Inc. Tgf-beta inhibitors
US10822337B2 (en) 2015-04-01 2020-11-03 Rigel Pharmaceuticals, Inc. TGF-β inhibitorC
MX2017013267A (es) 2015-04-15 2018-08-15 Regeneron Pharma Metodos para aumentar la fuerza y funcionalidad con inhibidores del factor de diferenciacion y crecimiento 8 (gdf8).
CN113896789A (zh) 2015-09-15 2022-01-07 供石公司 抗-原肌生长抑制素/潜伏肌生长抑制素抗体及其用途
SG10201707267RA (en) * 2015-12-18 2017-10-30 Chugai Pharmaceutical Co Ltd Anti-myostatin antibodies, polypeptides containing variant fc regions, and methods of use
JP7141336B2 (ja) 2015-12-25 2022-09-22 中外製薬株式会社 抗ミオスタチン抗体および使用方法
AU2017206069A1 (en) * 2016-01-08 2018-07-19 Scholar Rock, Inc. Anti-pro/latent myostatin antibodies and methods of use thereof
PT3368069T (pt) 2016-06-13 2020-11-11 Scholar Rock Inc Uso de inibidores da miostatina e terapias de combinação
KR102306744B1 (ko) 2016-06-17 2021-09-28 추가이 세이야쿠 가부시키가이샤 항-마이오스타틴 항체 및 사용 방법
CN109689099B (zh) 2016-08-05 2023-02-28 中外制药株式会社 用于预防或治疗il-8相关疾病的组合物
EP4218817A3 (en) 2017-01-06 2023-09-06 Scholar Rock, Inc. Methods for treating metabolic diseases by inhibiting myostatin activation
MA52417A (fr) 2018-03-01 2021-01-06 Regeneron Pharma Procédés de modification de composition corporelle
EP3898672A1 (en) 2018-12-18 2021-10-27 Regeneron Pharmaceuticals, Inc. Compositions and methods for enhancing body weight and lean muscle mass using antagonists against leptin receptor, gdf8 and activin a
WO2020132647A1 (en) 2018-12-21 2020-06-25 Northwestern University Use of annexins in preventing and treating muscle membrane injury
US20220062299A1 (en) 2018-12-26 2022-03-03 Northwestern University Use of glucocorticoid steroids in preventing and treating conditions of muscle wasting, aging and metabolic disorder
WO2024064842A1 (en) 2022-09-21 2024-03-28 Regeneron Pharmaceuticals, Inc. Methods of treating obesity, diabetes, and liver dysfunction

Family Cites Families (61)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4179337A (en) 1973-07-20 1979-12-18 Davis Frank F Non-immunogenic polypeptides
JPS6023084B2 (ja) 1979-07-11 1985-06-05 味の素株式会社 代用血液
US4554101A (en) 1981-01-09 1985-11-19 New York Blood Center, Inc. Identification and preparation of epitopes on antigens and allergens on the basis of hydrophilicity
US4640835A (en) 1981-10-30 1987-02-03 Nippon Chemiphar Company, Ltd. Plasminogen activator derivatives
JPS5896026A (ja) 1981-10-30 1983-06-07 Nippon Chemiphar Co Ltd 新規ウロキナ−ゼ誘導体およびその製造法ならびにそれを含有する血栓溶解剤
DE3380726D1 (en) 1982-06-24 1989-11-23 Japan Chem Res Long-acting composition
US4522811A (en) 1982-07-08 1985-06-11 Syntex (U.S.A.) Inc. Serial injection of muramyldipeptides and liposomes enhances the anti-infective activity of muramyldipeptides
US4816567A (en) 1983-04-08 1989-03-28 Genentech, Inc. Recombinant immunoglobin preparations
US4496689A (en) 1983-12-27 1985-01-29 Miles Laboratories, Inc. Covalently attached complex of alpha-1-proteinase inhibitor with a water soluble polymer
DE3675588D1 (de) 1985-06-19 1990-12-20 Ajinomoto Kk Haemoglobin, das an ein poly(alkenylenoxid) gebunden ist.
US4766106A (en) 1985-06-26 1988-08-23 Cetus Corporation Solubilization of proteins for pharmaceutical compositions using polymer conjugation
EP0272253A4 (en) 1986-03-07 1990-02-05 Massachusetts Inst Technology METHOD FOR IMPROVING GLYCOPROTE INSTABILITY.
US4791192A (en) 1986-06-26 1988-12-13 Takeda Chemical Industries, Ltd. Chemically modified protein with polyethyleneglycol
US4950221A (en) * 1986-07-18 1990-08-21 Gordon Robert T Process for affecting molecules in tissue
US5308752A (en) * 1990-05-23 1994-05-03 Univ. Of Iowa Research Foundation Diagnosis of autosomal muscular dystrophy
US6162896A (en) 1991-05-10 2000-12-19 The Salk Institute For Biological Studies Recombinant vertebrate activin receptors
DK0590058T3 (da) 1991-06-14 2004-03-29 Genentech Inc Humaniseret heregulin-antistof
US20030074680A1 (en) 1993-03-19 2003-04-17 Johns Hopkins University School Of Medicine Growth differentiation factor-8
US5994618A (en) 1997-02-05 1999-11-30 Johns Hopkins University School Of Medicine Growth differentiation factor-8 transgenic mice
US6673534B1 (en) 1995-10-26 2004-01-06 The Johns Hopkins University School Of Medicine Methods for detection of mutations in myostatin variants
US6465239B1 (en) 1993-03-19 2002-10-15 The John Hopkins University School Of Medicine Growth differentiation factor-8 nucleic acid and polypeptides from aquatic species and non-human transgenic aquatic species
US6607884B1 (en) 1993-03-19 2003-08-19 The Johns Hopkins University School Of Medicine Methods of detecting growth differentiation factor-8
CA2157577C (en) * 1993-03-19 2009-11-17 Se-Jin Lee Growth differentiation factor-8
US7393682B1 (en) 1993-03-19 2008-07-01 The Johns Hopkins University School Of Medicine Polynucleotides encoding promyostatin polypeptides
DK1378572T3 (da) * 1993-05-12 2007-02-05 Genetics Inst Llc BMP-11-sammensætninger
WO1995005846A1 (en) 1993-08-26 1995-03-02 Genetics Institute, Inc. Neural regeneration using human bone morphogenetic proteins
US7332575B2 (en) 1994-03-18 2008-02-19 The Johns Hopkins University School Of Medicine Growth differentiation factor-8 nucleic acid and polypeptide from aquatic species, and transgenic aquatic species
ATE305036T1 (de) 1994-07-08 2005-10-15 Univ Johns Hopkins Med Wachstums-differenzierungsfaktor-11
US6008434A (en) 1994-07-08 1999-12-28 Johns Hopkins University School Of Medicine Growth differentiation factor-11 transgenic mice
DK0766745T3 (da) * 1995-04-08 2002-11-25 Lg Chemical Ltd Monoklonalt antistof, som er specifikt for humant 4-1BB samt cellelinje, som producerer dette
US5723125A (en) 1995-12-28 1998-03-03 Tanox Biosystems, Inc. Hybrid with interferon-alpha and an immunoglobulin Fc linked through a non-immunogenic peptide
US7393528B2 (en) * 1997-01-09 2008-07-01 Tvedten Stephen L Biological pesticide
AU6274298A (en) 1997-02-05 1998-08-25 Johns Hopkins University School Of Medicine, The Growth differentiation factor-8
PT2045322E (pt) 1997-07-14 2015-10-16 Université de Liège Musculatura dupla em mamíferos
US6656475B1 (en) * 1997-08-01 2003-12-02 The Johns Hopkins University School Of Medicine Growth differentiation factor receptors, agonists and antagonists thereof, and methods of using same
US6891082B2 (en) 1997-08-01 2005-05-10 The Johns Hopkins University School Of Medicine Transgenic non-human animals expressing a truncated activintype II receptor
US6696260B1 (en) 1997-08-01 2004-02-24 The Johns Hopkins University School Of Medicine Methods to identify growth differentiation factor (GDF) binding proteins
AU8666398A (en) 1997-08-01 1999-02-22 Johns Hopkins University School Of Medicine, The Methods to identify growth differentiation factor (gdf) receptors
AU1276399A (en) 1997-11-07 1999-05-31 Genetics Institute Inc. Neuronal uses of bmp-11
WO1999024618A1 (en) 1997-11-10 1999-05-20 The Johns Hopkins University School Of Medicine Methods for detection of mutations in myostatin variants
AU2586199A (en) * 1998-02-05 1999-08-23 Johns Hopkins University School Of Medicine, The Growth differentiation factor-8
US6369201B1 (en) 1998-02-19 2002-04-09 Metamorphix International, Inc. Myostatin multimers
US6004937A (en) 1998-03-09 1999-12-21 Genetics Institute, Inc. Use of follistatin to modulate growth and differentiation factor 8 [GDF-8] and bone morphogenic protein 11 [BMP-11]
DE69941116D1 (de) 1998-05-06 2009-08-27 Metamorphix Inc G von gdf-8
WO2000011163A1 (en) 1998-08-20 2000-03-02 Regeneron Pharmaceuticals, Inc. Dcr5, a bmp-binding protein, and applications thereof
JP2003517580A (ja) 1999-01-21 2003-05-27 メタモーフイクス・インコーポレーテツド 増殖分化因子インヒビター及びそれらの用途
AU778470B2 (en) 1999-07-20 2004-12-09 Pharmexa A/S Method for down-regulating GDF-8 activity
AU2001241817A1 (en) 2000-02-29 2001-09-12 Zymogenetics Inc. Kunitz domain polypeptide zkun8
TW200526779A (en) 2001-02-08 2005-08-16 Wyeth Corp Modified and stabilized GDF propeptides and uses thereof
US7320789B2 (en) 2001-09-26 2008-01-22 Wyeth Antibody inhibitors of GDF-8 and uses thereof
MXPA04008150A (es) 2002-02-21 2005-06-17 Wyeth Corp Gasp1: una proteina que contiene dominio de folistatina.
KR20040096592A (ko) 2002-02-21 2004-11-16 와이어쓰 폴리스타틴 도메인을 포함하는 단백질
US20040138118A1 (en) 2002-09-16 2004-07-15 Neil Wolfman Metalloprotease activation of myostatin, and methods of modulating myostatin activity
US6696367B1 (en) 2002-09-27 2004-02-24 Asm America, Inc. System for the improved handling of wafers within a process tool
US7261893B2 (en) 2002-10-22 2007-08-28 Wyeth Neutralizing antibodies against GDF-8 and uses therefor
US20040223966A1 (en) 2002-10-25 2004-11-11 Wolfman Neil M. ActRIIB fusion polypeptides and uses therefor
US7511012B2 (en) 2002-12-20 2009-03-31 Amgen Inc. Myostatin binding agents
CA2526669A1 (en) 2003-06-02 2004-12-16 Wyeth Use of myostatin (gdf8) inhibitors in conjunction with corticosteroids for treating neuromuscular disorders
US7261717B2 (en) * 2003-09-11 2007-08-28 Skeletal Kinetics Llc Methods and devices for delivering orthopedic cements to a target bone site
MX2007011400A (es) 2005-03-23 2007-10-11 Wyeth Corp Deteccion de agentes moduladores de crecimiento y la diferenciacion del factor 8 (gdf 8).
US20060240488A1 (en) 2005-03-23 2006-10-26 Nowak John A Detection of an immune response to GDF-8 modulating agents

Also Published As

Publication number Publication date
ES2481165T3 (es) 2014-07-29
CA2469230A1 (en) 2003-04-03
US20110020330A1 (en) 2011-01-27
CA2469230C (en) 2017-02-21
EP1438068A2 (en) 2004-07-21
US7320789B2 (en) 2008-01-22
US20030138422A1 (en) 2003-07-24
US20120107928A1 (en) 2012-05-03
US20150024484A1 (en) 2015-01-22
BR0212809A (pt) 2005-12-13
EP1438068A4 (en) 2005-11-16
US7731961B1 (en) 2010-06-08
US9505831B2 (en) 2016-11-29
JP4452077B2 (ja) 2010-04-21
WO2003027248A3 (en) 2003-05-30
US8710202B2 (en) 2014-04-29
ZA200402265B (en) 2007-03-28
ZA200606082B (en) 2008-10-29
NZ532034A (en) 2006-06-30
MXPA04002834A (es) 2005-09-28
JP2009067798A (ja) 2009-04-02
DK1438068T3 (da) 2014-06-30
NO20041582L (no) 2004-04-19
PT1438068E (pt) 2014-07-24
WO2003027248A2 (en) 2003-04-03
US8092798B2 (en) 2012-01-10
HK1064284A1 (zh) 2005-01-28
EP2316851A1 (en) 2011-05-04
NO336528B1 (no) 2015-09-21
WO2003027248A8 (en) 2004-04-22
PL374221A1 (en) 2005-10-03
JP2005510212A (ja) 2005-04-21
AU2002347773B2 (en) 2008-03-13

Similar Documents

Publication Publication Date Title
EP1438068B1 (en) Antibody inhibitors of gdf-8 and uses thereof
AU2002347773A1 (en) Antibody inhibitors of GDF-8 and uses thereof
JP6960485B2 (ja) 線維芽増殖因子受容体２に対するモノクローナル抗体
RU2360925C2 (ru) Нейтрализующие антитела против gdf-8 и их применение
EP1572961B1 (en) Actriib fusion polypeptides and uses therefor
JP2019510739A (ja) Ｇｆｒａｌ受容体療法

Legal Events

Date Code Title Description
PUAI Public reference made under article 153(3) epc to a published international application that has entered the european phase

Free format text: ORIGINAL CODE: 0009012

17P Request for examination filed

Effective date: 20040405

AK Designated contracting states

Kind code of ref document: A2

Designated state(s): AT BE BG CH CY CZ DE DK EE ES FI FR GB GR IE IT LI LU MC NL PT SE SK TR

AX Request for extension of the european patent

Extension state: AL LT LV MK RO SI

REG Reference to a national code

Ref country code: HK

Ref legal event code: DE

Ref document number: 1064284

Country of ref document: HK

A4 Supplementary search report drawn up and despatched

Effective date: 20051005

RIC1 Information provided on ipc code assigned before grant

Ipc: 7C 07K 16/22 B

Ipc: 7C 07K 14/475 B

Ipc: 7C 07K 16/00 A

Ipc: 7A 61K 39/395 B

RAP1 Party data changed (applicant data changed or rights of an application transferred)

Owner name: WYETH LLC

GRAP Despatch of communication of intention to grant a patent

Free format text: ORIGINAL CODE: EPIDOSNIGR1

INTG Intention to grant announced

Effective date: 20131125

RAP1 Party data changed (applicant data changed or rights of an application transferred)

Owner name: WYETH LLC

GRAP Despatch of communication of intention to grant a patent

Free format text: ORIGINAL CODE: EPIDOSNIGR1

GRAS Grant fee paid

Free format text: ORIGINAL CODE: EPIDOSNIGR3

INTG Intention to grant announced

Effective date: 20140319

GRAA (expected) grant

Free format text: ORIGINAL CODE: 0009210

AK Designated contracting states

Kind code of ref document: B1

Designated state(s): AT BE BG CH CY CZ DE DK EE ES FI FR GB GR IE IT LI LU MC NL PT SE SK TR

REG Reference to a national code

Ref country code: GB

Ref legal event code: FG4D

REG Reference to a national code

Ref country code: CH

Ref legal event code: EP

Ref country code: CH

Ref legal event code: NV

Representative=s name: E. BLUM AND CO. AG PATENT- UND MARKENANWAELTE , CH

REG Reference to a national code

Ref country code: AT

Ref legal event code: REF

Ref document number: 669567

Country of ref document: AT

Kind code of ref document: T

Effective date: 20140615

REG Reference to a national code

Ref country code: IE

Ref legal event code: FG4D

REG Reference to a national code

Ref country code: DK

Ref legal event code: T3

Effective date: 20140626

REG Reference to a national code

Ref country code: DE

Ref legal event code: R096

Ref document number: 60246307

Country of ref document: DE

Effective date: 20140703

REG Reference to a national code

Ref country code: SE

Ref legal event code: TRGR

REG Reference to a national code

Ref country code: PT

Ref legal event code: SC4A

Free format text: AVAILABILITY OF NATIONAL TRANSLATION

Effective date: 20140714

REG Reference to a national code

Ref country code: ES

Ref legal event code: FG2A

Ref document number: 2481165

Country of ref document: ES

Kind code of ref document: T3

Effective date: 20140729

REG Reference to a national code

Ref country code: NL

Ref legal event code: T3

REG Reference to a national code

Ref country code: GR

Ref legal event code: EP

Ref document number: 20140401265

Country of ref document: GR

Effective date: 20140718

REG Reference to a national code

Ref country code: HK

Ref legal event code: GR

Ref document number: 1064284

Country of ref document: HK

Ref country code: SK

Ref legal event code: T3

Ref document number: E 16757

Country of ref document: SK

PGFP Annual fee paid to national office [announced via postgrant information from national office to epo]

Ref country code: TR

Payment date: 20140715

Year of fee payment: 13

PG25 Lapsed in a contracting state [announced via postgrant information from national office to epo]

Ref country code: EE

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20140521

REG Reference to a national code

Ref country code: DE

Ref legal event code: R097

Ref document number: 60246307

Country of ref document: DE

PLBE No opposition filed within time limit

Free format text: ORIGINAL CODE: 0009261

STAA Information on the status of an ep patent application or granted ep patent

Free format text: STATUS: NO OPPOSITION FILED WITHIN TIME LIMIT

26N No opposition filed

Effective date: 20150224

PG25 Lapsed in a contracting state [announced via postgrant information from national office to epo]

Ref country code: MC

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20140521

REG Reference to a national code

Ref country code: DE

Ref legal event code: R097

Ref document number: 60246307

Country of ref document: DE

Effective date: 20150224

PG25 Lapsed in a contracting state [announced via postgrant information from national office to epo]

Ref country code: BE

Free format text: LAPSE BECAUSE OF NON-PAYMENT OF DUE FEES

Effective date: 20140930

PGFP Annual fee paid to national office [announced via postgrant information from national office to epo]

Ref country code: BG

Payment date: 20150626

Year of fee payment: 14

PGFP Annual fee paid to national office [announced via postgrant information from national office to epo]

Ref country code: CZ

Payment date: 20150910

Year of fee payment: 14

Ref country code: DK

Payment date: 20150826

Year of fee payment: 14

Ref country code: FI

Payment date: 20150907

Year of fee payment: 14

Ref country code: PT

Payment date: 20150826

Year of fee payment: 14

Ref country code: CH

Payment date: 20150724

Year of fee payment: 14

Ref country code: SK

Payment date: 20150828

Year of fee payment: 14

PGFP Annual fee paid to national office [announced via postgrant information from national office to epo]

Ref country code: LU

Payment date: 20151007

Year of fee payment: 14

Ref country code: BE

Payment date: 20150916

Year of fee payment: 14

Ref country code: AT

Payment date: 20150825

Year of fee payment: 14

Ref country code: GR

Payment date: 20150828

Year of fee payment: 14

Ref country code: SE

Payment date: 20150908

Year of fee payment: 14

PGFP Annual fee paid to national office [announced via postgrant information from national office to epo]

Ref country code: NL

Payment date: 20150930

Year of fee payment: 14

PG25 Lapsed in a contracting state [announced via postgrant information from national office to epo]

Ref country code: CY

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20140521

REG Reference to a national code

Ref country code: FR

Ref legal event code: PLFP

Year of fee payment: 15

PG25 Lapsed in a contracting state [announced via postgrant information from national office to epo]

Ref country code: BE

Free format text: LAPSE BECAUSE OF NON-PAYMENT OF DUE FEES

Effective date: 20160930

REG Reference to a national code

Ref country code: DK

Ref legal event code: EBP

Effective date: 20160930

PG25 Lapsed in a contracting state [announced via postgrant information from national office to epo]

Ref country code: SE

Free format text: LAPSE BECAUSE OF NON-PAYMENT OF DUE FEES

Effective date: 20160927

Ref country code: FI

Free format text: LAPSE BECAUSE OF NON-PAYMENT OF DUE FEES

Effective date: 20160926

REG Reference to a national code

Ref country code: CH

Ref legal event code: PL

REG Reference to a national code

Ref country code: SE

Ref legal event code: EUG

REG Reference to a national code

Ref country code: NL

Ref legal event code: MM

Effective date: 20161001

REG Reference to a national code

Ref country code: AT

Ref legal event code: MM01

Ref document number: 669567

Country of ref document: AT

Kind code of ref document: T

Effective date: 20160926

PG25 Lapsed in a contracting state [announced via postgrant information from national office to epo]

Ref country code: PT

Free format text: LAPSE BECAUSE OF NON-PAYMENT OF DUE FEES

Effective date: 20170327

Ref country code: CZ

Free format text: LAPSE BECAUSE OF NON-PAYMENT OF DUE FEES

Effective date: 20160926

REG Reference to a national code

Ref country code: SK

Ref legal event code: MM4A

Ref document number: E 16757

Country of ref document: SK

Effective date: 20160926

PG25 Lapsed in a contracting state [announced via postgrant information from national office to epo]

Ref country code: NL

Free format text: LAPSE BECAUSE OF NON-PAYMENT OF DUE FEES

Effective date: 20161001

REG Reference to a national code

Ref country code: GR

Ref legal event code: ML

Ref document number: 20140401265

Country of ref document: GR

Effective date: 20170411

PG25 Lapsed in a contracting state [announced via postgrant information from national office to epo]

Ref country code: GR

Free format text: LAPSE BECAUSE OF NON-PAYMENT OF DUE FEES

Effective date: 20170411

Ref country code: CH

Free format text: LAPSE BECAUSE OF NON-PAYMENT OF DUE FEES

Effective date: 20160930

Ref country code: LI

Free format text: LAPSE BECAUSE OF NON-PAYMENT OF DUE FEES

Effective date: 20160930

Ref country code: SK

Free format text: LAPSE BECAUSE OF NON-PAYMENT OF DUE FEES

Effective date: 20160926

REG Reference to a national code

Ref country code: FR

Ref legal event code: PLFP

Year of fee payment: 16

PG25 Lapsed in a contracting state [announced via postgrant information from national office to epo]

Ref country code: AT

Free format text: LAPSE BECAUSE OF NON-PAYMENT OF DUE FEES

Effective date: 20160926

Ref country code: LU

Free format text: LAPSE BECAUSE OF NON-PAYMENT OF DUE FEES

Effective date: 20160926

Ref country code: BG

Free format text: LAPSE BECAUSE OF NON-PAYMENT OF DUE FEES

Effective date: 20170406

REG Reference to a national code

Ref country code: BE

Ref legal event code: MM

Effective date: 20160930

PG25 Lapsed in a contracting state [announced via postgrant information from national office to epo]

Ref country code: DK

Free format text: LAPSE BECAUSE OF NON-PAYMENT OF DUE FEES

Effective date: 20160930

REG Reference to a national code

Ref country code: FR

Ref legal event code: PLFP

Year of fee payment: 17

PGFP Annual fee paid to national office [announced via postgrant information from national office to epo]

Ref country code: FR

Payment date: 20200814

Year of fee payment: 19

Ref country code: DE

Payment date: 20200812

Year of fee payment: 19

Ref country code: IE

Payment date: 20200826

Year of fee payment: 19

Ref country code: GB

Payment date: 20200828

Year of fee payment: 19

PGFP Annual fee paid to national office [announced via postgrant information from national office to epo]

Ref country code: IT

Payment date: 20200921

Year of fee payment: 19

PGFP Annual fee paid to national office [announced via postgrant information from national office to epo]

Ref country code: ES

Payment date: 20201001

Year of fee payment: 19

REG Reference to a national code

Ref country code: DE

Ref legal event code: R119

Ref document number: 60246307

Country of ref document: DE

GBPC Gb: european patent ceased through non-payment of renewal fee

Effective date: 20210926

PG25 Lapsed in a contracting state [announced via postgrant information from national office to epo]

Ref country code: TR

Free format text: LAPSE BECAUSE OF NON-PAYMENT OF DUE FEES

Effective date: 20160926

PG25 Lapsed in a contracting state [announced via postgrant information from national office to epo]

Ref country code: IE

Free format text: LAPSE BECAUSE OF NON-PAYMENT OF DUE FEES

Effective date: 20210926

Ref country code: GB

Free format text: LAPSE BECAUSE OF NON-PAYMENT OF DUE FEES

Effective date: 20210926

Ref country code: FR

Free format text: LAPSE BECAUSE OF NON-PAYMENT OF DUE FEES

Effective date: 20210930

Ref country code: DE

Free format text: LAPSE BECAUSE OF NON-PAYMENT OF DUE FEES

Effective date: 20220401

PG25 Lapsed in a contracting state [announced via postgrant information from national office to epo]

Ref country code: IT

Free format text: LAPSE BECAUSE OF NON-PAYMENT OF DUE FEES

Effective date: 20210926

REG Reference to a national code

Ref country code: ES

Ref legal event code: FD2A

Effective date: 20221031

PG25 Lapsed in a contracting state [announced via postgrant information from national office to epo]

Ref country code: ES

Free format text: LAPSE BECAUSE OF NON-PAYMENT OF DUE FEES

Effective date: 20210927